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amniotic fluid analysis


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amniotic fluid analysis

  2. 2. Utilities of Analysis:• Determination of fetal lung maturity• Detection of fetal distress• Cytogenetic analysis• Detection of hereditary, teratology and infectious disorders• Determination of fetal age
  3. 3. Formation and Physiology:• formation in the amnion is regulated by balance between the production of fetal urine and lung fluid and the absorption from fetal swallowing and intramembranous flow• Functions: (1) provides a protective cushion for the fetus (2) allows fetal movement (3) stabilizes the temperature to protect the fetus from extreme temperature changes (4) permit proper lung development.
  4. 4. Volume: Approximately 35 mL during the 1st trimester, peaks during the 3rd trimester (approx.1 L) and gradually decreases prior to deliver; major contributors are maternal circulation (1st trimester) and fetal urine (after the 1st trimester)Polyhydramnios Excessive accumulation of amniotic fluid usually resulting from the failure of the fetus to begin swallowing; indicates fetal distress often associated with neural tube disorders
  5. 5. Oligohydramnios Decreased amniotic fluid due primarily to increased fetal swallowing urinary tract deformities, and membrane leakageComposition: Same as that of maternal plasma plus a small amount of sloughed fetal cells, biochemical substance produced by the fetus, and a portion from the fetal respiratory tract, fetal urine, the amniotic membrane, and the umbilical cod
  6. 6. 1. Specimen Considerationsa. Amniocentesis • Needle aspiration of amniotic fluid from the amniotic as; may be transabdominal or transvaginal; safety performed after the 14th week of gestation , volume collected:
  7. 7. 2. Specimen handlinga. Fluid for FLM tests • Transported in ice and refrigerated up to 72 hours prior to testing or kept frozen and tested within 72 hours; filtration or low-speed centrifugation is recommendedb. Fluid for cytogenetic studies • Maintained at room temperature or incubated at 37°C prior to analysis
  8. 8. c. Fluid for chemical testing • Separated from cellular elements and debris ASAP to prevent distortion of chemical constituents by cellular metabolism or disitegration.d. Fluid for bilirubin analysis • placed in amber bottles or containers covered with a black plastic.
  9. 9. Gross Examination Appearance Significance Colorless with Normal slight to moderate turbidity Blood- streaked Traumatic tap, abdominal trauma,intra-amniotic hemorrhage Yellow HDN dark- green Meconium Dark red- brown Fetal Death
  10. 10. Tests for Lung Maturity1. Lecithin/ sphingomyelin ratioa. Method:____________________________b. Principles: Lecithin is produced at a relatively low and constant rate until the 35th week of gestation while sphingomyelin is produced at a constant rate after about 26 week’ gestation and therefore conserve as a control on which to base the rise in lecithin. Prior to 35 week’ gestation, L/S ratio is ˂1.6 and rises to >2.0 when lecithin production increases.2. Amniostat-FLMa. Method:________________________b. Principle: the test uses antisera for phosphatidly glycerol and is affectected by specimen contamination with blood and meconium.
  11. 11. 3. Foam stability index a. method:_____________________________ b. Principle: a semiquatitative measure of the amount of surfactant is done by adding 0.5 mL of amniotic fluid to increasing amounts of 95% ethanol (0.42 mL to 0.55 mL in 0.01-mL increments), shaken for 15 seconds, and allowed to sit undisturbed for 15 minutes. If a sufficient amount of phospholipid is present, a continuous line of bubbles will be observed even in the presence of alcohol, an anti-foaming agent.4. Microviscosity a. Method:______________________ b. Principle: Phospholipids decrease the microviscosity of amniotic fluid and the change is detected by determining the surfactant to albumin ratio (mg/g) based on the polarization of a fluorescent dye that combines (internal standard, decreased fluorescence lifetime and high polarization).
  12. 12. 5. Lamellar body count a. Method:___________________________ b. Principle: Lamellar bodies (lamellated phospholipids that represent a storage from of surfactants secreted by the type II pneumocytes of the fetal lung)range in size from 1.7 to 7.3 fL, and therefore can be counted using the platelet channel of hematology analyzers.6. Optical density at 650 nm a. Method:_________________________ b. Principle : the increase in OD of the amniotic fluid caused by the presence of lamella bodies in determined by centrifuging the specimen at 2000 g for 10 min and reading the absorbance at 650 nm.
  13. 13. Table 24. Tests for fetal lung maturity Normal Significance valuesL/S ratio ≥2.0 FLMAmniostat-FLM Positive FLM/phosphotidyl glycerolFoams Stability index ≥47 FLMMicroviscosity ≥55 mg/ g FLMLamellar body count ≥32,00/ mL FLMOD at 650 nm ≥0.150 FLMBilirubin scan A 450 less HDN .025Alpha Fetoprotein Less than Neural tube disorder 2.0 MoM
  14. 14. Test for Fetal Distress 1. Bilirubin assay a. Method: _____________________ b. Principle: the optical density of amniotic fluid is normally highest at 365 nm and decreases linearly to 550 nm except when bilirubin is present where a rise in OD is seen at 450 nm. The ᴧ ᴧ450 is then plotted on a liley graph to determine the severity of HDN and the need for interventions. 2. Alpha fetoprotein a. Method:_________________ b. Principle: The Test is based on the measurement of the neural tube defects using an automated immunoassay method: results are reported in terms of multiples of the median with a value >2 MoM considered abnormal
  15. 15. 3. Acetylcholinesterase a. Method: ____________________ b. Principle: Ache is an enzyme derived primarily from the neural tissue and is normally absent in amniotic fluid. Its presence in amniotic fluid in conjunction with elevated AFP values is highly diagnostic of NTDs.
  16. 16. Other Tests 1. Differentiation of amniotic fluid from maternal urine • Creatinine is ˂3.5 mg/ dL and urea is ˂30 mg/dL in amniotic fluid, whereas high as 10 mg/ dL creatinine and 300 mg/dL urea may be found in urine 2. Determination of fetal age • AF creatinine level ranges from 1.5 to 2.0 mg/ dL prior to 36 weeks’ gestation and rises above 2.0 mg/dL thereafter, providing a means of determining fetal age as >36 weeks 3. Kleihauer-Betke test • used to determine the source of the blood (maternal or fetal) in a bloody specimen for further case management.