1. Amniotic fluid
Lec. 18

2. Testing amniotic fluid associated with cytogeneic analysis.
Amniotic fluid is a product of fetal metabolism which provide an information about fetal maturation.

4. Physiology of amniotic fluid
Amniotic fluid is present in the amnion a membranous sac that surrounds the fetus to provide a protective cushion for the fetus and allow the movement

5. Exchanges of water and chemicals takes place between the fluid, the fetus and the maternal circulation.
Amniotic fluid increases during pregnancy which reach about 1L during the 3rd trimester.
It decreases prior delivery.
Its composition is similar to that of the maternal plasma and contains a small amount of sloughed fetal cells.

6. LIST THE COMPOSITION OF AMNIOTIC FLUID
                               
(AMNIOTIC FLUID)                                (SERUM)
Color                            colorless to straw                                        (straw) Calcium                             4.0 mEq/L                                     ( mEq/L) Chloride                          mEq/L                                   ( mEq/L) CO2                                  16.0 mEq/L                                      ( mEq/L) Creatinine                         1.8 mg/dL                                       ( mg/dL) Glucose                             29.8 mg/dL                                     ( mg/dL) pH                                          7.04                                                   ( ) Potassium                         4.9 mEq/L                                      ( mEq/L) Sodium                             mEq/L                                  ( mEq/L) Total Protein                      2.5 gm/dL                                      ( gm/dL) Albumin                              1.4 gm/dL                                       ( gm/dL) Urea                                    31.0 mg/dL                                    ( gm/dL) Uric Acid                             4.9 mg/dL                                      ( mg/dL

7. After the 1st trimester, fetal urine is the major donor to the amniotic fluid volume.
When fetal urine production occurs the fetal swallowing of the amniotic fluid to regulate the increase in fluid from fetus urine.
Hydramnios is an indication of fetal distress when the fetus fail in swallowing and cause excessive accumulation of amniotic fluid.

9. Oligohydromnios decreased amniotic fluid which is due to increase fetal swallowing or membrane leakage.
Chemical composition changes when fetal urine begins.
The conc. of creatinine, urea, and uric acid ↑↑ while the conc. of glucose and protein↓↓
Conc. of electrolytes, enzymes, hormones and metabolic end product vary.

10. Measurment of amniotic fluid
Creatinine determine the fetal age.
Differentiation between amniotic fluid and maternal urine determine the possible premature rupture during specimen collection.
Levels of creatinine and urea are lower in amniotic fluid than that in urine.
Measurement of glucose and protein is less reliable indicator.

11. Specimen collection Amniocentesis
Amniocentesis is used to determine the health of an unborn baby. Amniotic fluid contains cells that are normally shed from the fetus (after 14 weeks).
Samples of these cells are obtained by withdrawing some amniotic fluid. The chromosome analysis (16 weeks) of these cells can be performed to determine abnormalities.

13. Fluid for bilirubin analysis in case of hemolytic disease of the newborn (HDN) must kept in amber colored tubes.
Fluid for fetal lung maturity (FLM) should placed in ice and refrigerated prior to testing.
All fluid should be separated from cellular elements to prevent distortion of chemical constituents by cellular metabolism.
Separation by centrifugation or filtration but filtration is recommended to prevent loss of phospholipids.

14. Color and appearance
Normally is colorless and may exhibit slight to moderate turbidity from cellular debris.
The presence of bilirubin give the fluid yellow color
Meconium is a gelatinous mixture of secretions from the intestinal tract of the fetus. It is varying shades of green, dependent upon the amount of biliverdin present.
A very dark red-brown fluid associated fetal death.

15. Test for fetal distress
Hemolytic disease of the newborn (HDN)
When anti-Rh antibodies present in the maternal circulation cross the placenta, it results in the appearance of the red blood cell degradation which produce bilirubin in the amniotic fluid.
By measuring the amount of bilirubin the degree of hemolysis may be determine.

16. Measurment is by spectrophotometic analysis (365nm and 550nm).
When bilirubin is present a rise of OD will be seen at 450nm.
Specimens must kept in amber colored tubes and care must be taken to reduce contamination with hemoglobin, meconium and other debris which affect the analysis.

17. Neural tube deffects
Increased level of Alpha-fetoprotein (AFP) (a protein produced by the fetal liver) in both maternal circulation and AF indicating a neural tube defects.
Neural tube defects affect the baby's spinal cord and brain. Anencephaly and spinal bifida are the two most common neural tube defects.

18. Neural Tube Defects

19. Increased levels are found in the maternal serum and AF when the skin fails to close over the neural tissue.
The risk for an open NTD or other fetal abnormality depends on the degree of elevation in the AFAFP. The results of amniotic fluid acetylcholinesterase testing and other significant risk factor.

20. Acetylcholinesterase (AChE) Assay - Acetylcholinesterase (AChE) is a neural enzyme present in cerebral spinal fluid and fetal blood. AChE is not present in maternal blood and is not normally detectable in amniotic fluid. The abnormal presence of acetylcholinesterase in amniotic fluid is suggestive of an open fetal defect.

21. Test for fetal Maturity Fetal lung maturity
Respiratory stress is the most frequent complication of early delivery.
The test determine the maturity of the fetal lungs FLM.

22. Lecithin- Sphingomyelin Ratio
Lecithin- Sphingomyelin (L/S) ratio.
Lecithin is the primary component of the surfactents (phospholipids, neutral lipids and proteins) which make up the alveolar lining and account for alveolar stability.
Lethithin ↓ until 35th week of gestation and then it will ↑ to stabiles the fetal lung alveoli.

23. Sphingomyelin is a lipid at constant rate after ~26 weeks of gestation.
Lecithin and sphingomyelin both presence in AF.
Measurment of Lecithin and sphingomyelin by using thin layer chromatography.

24. Amniostat-FLM Foam stability
Phosphatidyl glycerol is essential for lung maturity.
It is parallels that of lecithin (its production is delayed in diabetic mother).
Foam stability
Mechanical screening test called the foam or shake test.
The presence of bubbles indicates that a sufficient amount of phospholipid.