Amniotic fluid analysis

• Amniotic fluid


  • Formed from fetal cell metabolism and fetal urine
  • Found in the amnion
  • Contributes to approximately 35 ml of amniotic fluid during the 1st trimester of pregnancy
  • Fetal urine is the major contributor to amniotic fluid volume after the 1st trimester of pregnancy
• Functions of amniotic fluid


  • Protective cushion for the fetus
  • Allows fetal movement
  • Protects fetus from extreme temperature changes
  • Permit proper lung development
  • Allows exchanges of water and chemicals
• Amniocentesis


  1. Needle aspiration of amniotic fluid

  2. Safely done after the 14th week of gestation

  3. Possible accidental bladder puncture; cause contaminated amniotic fluid by maternal urine
• Amniotic fluid collection timing


  • 2nd trimester (15th - 18th week of gestation): assessment of genetic defects and intrauterine growth restrictions
  • 3rd trimester (20th - 24th week of gestation): assessment of fetal pulmonary maturity or fetal hemolytic disease
• Maximum amount of amniotic fluid aspirated is 30 ml
• First 2-3 ml of amniotic fluid is discarded due to possible contamination
• Specimen considerations


  • Bilirubin analysis: specimens must be protected from light
  • Fetal lung maturity testing: placed on ice for delivery and refrigerated prior to testing, low speed centrifugation for no longer than 5 minutes (500 to 1000 g) to prevent loss of phospholipids
  • Cytogenetic studies: specimens maintained at room or body temperature to prolong cells' viability
  • Chemical testing: fluid must be separated from cells and cellular debris as soon as possible
• Variations in amniotic fluid color


  • Blood-streaked: Traumatic tap, abdominal trauma, intra-amniotic hemorrhage
  • Yellow: Hemolytic disease of the newborn
  • Dark-green: Meconium
  • Dark red-brown: Fetal death
• Hydramnios


  Increased amniotic fluid volume, due to decreased fetal swallowing of urine; possible neural tube defect
• Oligohydramnios


  Decreased amniotic fluid volume, may be due to increased fetal swallowing of urine, urinary tract deformities or membrane leakage
• During the 1st trimester, the amniotic fluid volume is approximately 35 ml, primarily supplied by maternal circulation. However, by the 3rd trimester, this volume can increase around 800-1000 ml / 1 liter.
• L/S Ratio


  Reference method, lecithin is the primary component of the surfactant that maintains alveolar stability, sphingomyelin is measured to serve as control, cannot be used when contaminated with blood and meconium, a low L/S ratio (<1.5) can develop respiratory distress syndrome
• L/S Ratio values


  • 26th week: L<S
  • 36th week: L=S
  • After 36th week: L>S
  • Normal Value: >2.0
• Phosphatidylglycerol


  Surfactant, may be used in place of L/S ratio, production parallels that of lecithin but delayed in diabetic mothers
• Amniostat-FLM


  Serological test, uses antisera specific for phosphatidyl glycerol, not affected by blood and meconium contamination
• Foam Test


  Done by shaking amniotic fluid added with 95% ethanol for 15 seconds, presence of bubbles for 15 minutes is the positive result, qualitative method measure the significant amount of surfactant in amniotic fluid (specimen)
• Foam Stability Index


  Semi quantitative measure of the amount of surfactants present, fluid is made to react with various amounts of 95% ethanol, value of >47 indicates fetal lung maturity
• Microviscosity Assay


  Principle: presence of phospholipids decreases the microviscosity of amniotic fluid, this change in microviscosity is measured using fluorescence polarization by Abbott TDx analyzer, albumin is used as internal standard, value of ≥ 55 mg/g indicates fetal lung maturity
• Lamellar bodies


  Surfactants are secreted as lamellar bodies which enters the alveolar spaces and amniotic fluid, the number of lamellar bodies correlates to the amount of lungs surfactant and they are counted using resistance pulse counting (electrical impedance), value of >32,000/ml indicates fetal lung maturity, optical density can also be measured to assess presence of lamellar bodies, an OD of 0.150 correlated with a > 2.0 LS ratio and presence of phosphatidylglycerol
• Alpha-fetoprotein


  Screening test for Neural tube defects (NTD's), a major protein produced by the fetal liver during early gestation, found in maternal serum due to combined circulation and amniotic fluid due to fetal urination, normal values are based on gestational age with the peak level at 12-15 weeks of gestation, after which it will start to decline, both serum and amniotic levels are reported as multiples of the median
• Acetylcholinesterase


  Confirmatory test for Neural tube defects (NTD's), done after an elevated AFP level is detected to confirm neural tube defects, blood contamination will falsely increase the result
• Bilirubin analysis


  Done for the evaluation of Hemolytic disease of the newborn (HDN), bilirubin is measured using spectrophotometry and values are plotted on the Liley graph, oxyhemoglobin is the main inference in bilirubin measurement at OD450 and is removed through chloroform extraction
• Liley graph zones


  • Zone I: Normal or slightly affected
  • Zone II: Moderately affected, close monitoring, preparation for possible intervention
  • Zone III: Severely affected, requires intervention (induce labor, intrauterine transfusion)
• Summary of tests for fetal wellbeing and maturity


  • Bilirubin Scan: Δ𝐴!"# > 0.025 (Hemolytic disease of the newborn)
  • Alpha-fetoprotein: < 2.0 𝑀𝑜𝑀 (Neural tube disorders)
  • Lecithin-sphingomyelin ratio: ≥ 2.0 (Fetal lung maturity)
  • Amniostat - FLM: 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 (Fetal lung maturity / phosphatidylglycerol)
  • Foam stability index: ≥ 47 (Fetal lung maturity)
  • Microviscosity: ≥ 55 𝑚𝑔/𝑔 (Fetal lung maturity)
  • Optical density 650nm: ≥ 0.150 (Fetal lung maturity)
  • Lamellar body count: ≥ 32,000/𝜇𝑙 (Fetal lung maturity)
  • Creatinine: > 2𝑚𝑔/𝑑𝑙 (36th weeks of gestation) (Fetal maturity/Fetal age)
• Gastric fluid


  Hydrochloric acid (main acid), electrolytes, mucus (protects the gastric mucosa from acidity), pepsin (major digestive enzyme), gastrin, lipase (fat digestion), renin, LDH, AST, ALT and ribonuclease
• Patient should fast for 12-15 hours with no medication during the last 24 hours, and should avoid excessive swallowing of saliva during gastric fluid collection
• Gastric fluid collection methods


  • Intubation Method: gastric fluid is collected by inserting a gastric tube through the buccal or nasal cavity (Ewald's tubes, Rehfuss tubes, Sawyer tubes, Levine tubes, Miller abbott tubes)
  • Diagnex Blue Method: Tubeless method wherein the patient is made to swallow an ion exchange resin with a blue dye (Azure A)
• Variations in gastric fluid appearance


  • Yellow to Green: Regurgitation of bile into the stomach
  • Red: Blood from intubation trauma
  • Coffee ground: Old blood (gastritis, ulcer, carcinoma or swallowed from the mouth, nasopharynx, lungs)
• Normal gastric fluid volume


  • Fasting state: 20 - 50 mL
  • After a test meal: 20 - 80 ml
  • After chemical stimulation: 45 - 150 ml
• Variations in gastric fluid volume


  • Increased: Hypomotility, pyloric obstruction, ZE syndrome
  • Decreased: Gastric hypermotility
• Total acidity


  Normal total acidity: 40 to 70 mEq/L (HCI plus combined acids), free HCI: 20 to 40 mEq/L, free acidity: free HCI, organic acids and acid salts
• Gastric acidity conditions


  • Anacidity: failure of the stomach acidity to fall lower than 6.0 in a stimulation test
  • Hypochlorhydria: physiologic failure of pH to fall below 3.5, but decreases 1.0 in pH after stimulation
  • Achlorhydria: physiologic failure of pH to fall below 3.5 or 1.0 after stimulation
  • Euchlorhydria: normal gastric acidity
  • Achylia: absence of all acids
• Epithelial cells, starch granules, yeasts, bacteria, fat droplets, tissue debris, leukocytes, erythrocytes and food remnants are evaluated in the microscopic analysis of gastric fluid
• Basal Acid Output (BAO)


  Measured fasting levels of gastric production, collection during the 12-15 hour fasting state, four (15 or 30-minute interval) specimens are collected and volume, pH, titratable acidity and calculated acid output is determined, normal: 0 to 6 mEq/hour
• Maximal Acid Output (MAO)


  Involves stimulation of fluid formation through use of stimulants, acid collected in an hour after collection, normal: 5 to 40 mEq/hour
• Gastric stimulants


  • Histamine: exerts unpleasant systemic effects on blood vessels and smooth muscles, Histalog: Bomer of histamin with preferred effects on gastric acid collection
  • Pentagastrin: most preferred; synthetic analog of gastrin
  • Insulin: assess successful vagotomy procedure
• Test meals


  • Ewald's Meal: Bread and tea without sugar or water (routinely used; aka breakfast meal)
  • Boa's Meal: Oat meal (lactic acid determination)
  • Reigel's Meals: Beef steak and mashed potato (detection of achylia and hypoacidity)
  • Lavine's Meals (Alcohol Test Meal): Ethyl alcohol and methylene blue (alkali regurgitation to the stomach)
  • Sham Feeding: Sandwich is cleaved and spitted out (fictitious feeding)
• Gastric acid output conditions


  • Normal: BAO 2.5, MAO 25.0, BAO/MAO 10
  • Pernicious anemia: BAO 0, MAO 0, BAO/MAO 0
  • Duodenal ulcer: BAO increased, MAO increased
• Gastric stimulants


  1. Histamine: exerts unpleasant systemic effects on blood vessels and smooth muscles

  2. Histalog: Bomer of histamin with preferred effects on gastric acid collection

  3. Pentagastrin: most preferred; synthetic analog of gastrin

  4. Insulin: assess successful vagotomy procedure
• Test meals


  • Ewald's Meal: Bread and tea without sugar or water
  • Boa's Meal: Oat meal
  • Reigel's Meals: Beef steak and mashed potato
  • Lavine's Meals (Alcohol Test Meal): Ethyl alcohol and methylene blue
  • Sham Feeding: Sandwich is cleaved and spitted out