amniotic fluid analysis

1. AMNIOTIC FLUID

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. 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. Volume: Approximately 35 mL during the 1 st trimester, peaks during the 3 rd trimester (approx.1 L) and gradually decreases prior to deliver; major contributors are maternal circulation (1 st trimester) and fetal urine (after the 1 st 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. Oligohydramnios Decreased amniotic fluid due primarily to increased fetal swallowing urinary tract deformities, and membrane leakage Composition : 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. 1. Specimen Considerations a. Amniocentesis Needle aspiration of amniotic fluid from the amniotic as; may be transabdominal or transvaginal; safety performed after the 14 th week of gestation , volume collected :

7. 2. Specimen handling a. 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 recommended b. Fluid for cytogenetic studies Maintained at room temperature or incubated at 37°C prior to analysis

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. Gross Examination Appearance Significance Colorless with slight to moderate turbidity Normal Blood- streaked Traumatic tap, abdominal trauma,intra-amniotic hemorrhage Yellow HDN dark- green Meconium Dark red- brown Fetal Death

10. Tests for Lung Maturity 1. Lecithin/ sphingomyelin ratio Method:____________________________ Principles: Lecithin is produced at a relatively low and constant rate until the 35 th 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-FLM Method:________________________ Principle: the test uses antisera for phosphatidly glycerol and is affectected by specimen contamination with blood and meconium.

11. 3. Foam stability index method:_____________________________ 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 Method:______________________ 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. 5. Lamellar body count Method:___________________________ 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 Method:_________________________ 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. Table 24. Tests for fetal lung maturity Normal values Significance L/S ratio ≥2.0 FLM Amniostat-FLM Positive FLM/ phosphotidyl glycerol Foams Stability index ≥47 FLM Microviscosity ≥55 mg/ g FLM Lamellar body count ≥32,00/ mL FLM OD at 650 nm ≥0.150 FLM Bilirubin scan A 450 less .025 HDN Alpha Fetoprotein Less than 2.0 MoM Neural tube disorder

14. Test for Fetal Distress 1. Bilirubin assay Method: _____________________ 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 ᴬ450 is then plotted on a liley graph to determine the severity of HDN and the need for interventions. 2. Alpha fetoprotein Method:_________________ 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. 3. Acetylcholinesterase Method: ____________________ 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. 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.