AMNIOTIC FLUID ANALYSIS -Found in the membrane sac that surrounds the fetus and provides cushion to protect

fetus. Also provides movement for the fetus. - Formed from the metabolism of fetal cells, transfer of water across the placental membrane, and in the third trimester by fetal urine. - Amnion membranous sac that surrounds the fetus IMPORTANCE: 1. Determine fetal lung maturity 2. Monitor severity of hemolytic disease of the newborn 3. Determine the surfactant activity 4. Diagnosis of chronic fetal distress 5. Determine of prenatal infections 6. Determine of neural tube defects 7. For hormonal evaluation 8. Detect other genetic disorder During the first trimester approx. 35 ml derived from maternal circulation (maternal plasma) FLUID FORMATION: 1. Metabolism of fetal cells (fetal gastro intestinal, respiratory) 2. Transfer of water across the placental membrane 3. Fetal urine - major source during the third trimester. - Fetus begins swallowing the amniotic fluid in an amount equal to the urine output. Amniotic fluid volume = 500 to 1,000 ml at term - Increase cell metabolism and placental water exchanges are primarily responsible for bringing the total volume of term. CHARACTERISTICS: 1. Low specific gravity = about 1.008 2. Mildly alkaline pH = about 7.5 3. Electrolyte content is similar to maternal plasma 4. Calcium content is low = about 5.5 mg/dl. 5. Rate of exchange of fluid = 300-400 ml. 6. Volume varies according to the stage of pregnancy 10th week 30 ml. 20th week 350 ml. 38th week almost 1L. TERMINOLOGIES: Hydramnios- volume of > 2000 ml. - decreased fetal swallowing of fetal urine. - Neural tube defects in 2nd trimester. Oligohydramnios- volume of <50 ml. - increased in fetal swallowing of fetal urine - Urinary tract deformities - membrane leakage METHODS USED FOR SAMPLE COLLECTION: 1. Amniocentesis - procedure in obtaining amniotic fluid for analysis by needle aspiration into the amniotic sac. Advantages:

A. Procedure is relatively safe. B. Procedure can be obtained in an outpatient basis. C. Supernatant fluid can be analyzed simultaneously for Alpha-feto Protein concentration. 2. Chronic Villus Biopsy or Chorionic Villus Sampling - An alternate to amniocentesis - Carried out at 8-10 weeks (first trimester) for the diagnosis of genetic defects in the fetus. Advantages: a. Allows rapid cytogenetic analysis. b. Direct cell biochemical genetic test. c. Short term culture Disadvantages: a. Cell mosaicism condition in which patches of tissue of unlike genetic constitution are mingled together. b. Alpha-feto Protein analysis is not possible. INDICATIONS FOR AMNIOCENTESIS: 1. Second trimester amniocentesis - Carried out to diagnose genetic disease and developmental defects. - Early (14-16 weeks) cytogenetic analysis. - Undergoes amniotic cell culture. 2. Mid trimester amniocentesis - Carried out to detect bile pigment level in amniotic fluid, in hemolytic disease. - Fetal distress - Alpha feto Protein (AFP) to detect neural tube defect (NTD) skin fails to close and neural tissue is exposed. 3. Third trimester amniocentesis - Assess severity of erythroblastosis fetalis - predict neonatal respiratory distress (late 2nd, 3rd trimester) COMPLICATIONS OF AMNIOCENTESIS: a. Fetal or placental trauma- accidental hitting of fetus and placenta during needle aspiration. b. Hemorrhage c. Maternal alloimmunization- happens as a result of hemorrhage. d. Fluid leakage- causes decreased amniotic fluid (Oligohydramnios) e.Infection Test done on second trimester amniocentesis sample: 1. Cytogenetics (Cytogenetic Analysis) - Purpose early diagnosis of both numerical and structure chromosome disorder. - Method amniotic cell culture (growing fetal cells) - includes karyo typing (allows enumeration of chromosomes, detection of morphologic changes like translocation. Translocation would lead to mental retardation and other congenital malformation. 2. Cells biochemical genetics - Purpose: amniotic cells are used in order to diagnose a suspected 1. Inborn error of metabolism,

2. Specific genetic blood disorder 3. Sex linked disease 3. Fluid Supernate Analysis - Detect some genetic diseases using the amniotic supernate fluid. - Cystic fibrosis an autosomal recessive disorder that cause severe pulmonary disease, there is thick mucus that cause plugging of bronchi. 4. Fluid Discoloration- Detect any discoloration of the amniotic fluid that indicates fetal well being inside the mother. Normal color- straw and clear Green due to meconium Yellow due to increase bilirubin (HDN) Bloody red: Trauma Green or brown-indicate greater risk of miscarriage Dark brown or reddish brown: Fetal death -Brought about by the degradation products of intra uterine blood, Bilirubin -Turbidity of amniotic fluid indicates infection. 5. Amniotic AFP test- Purpose: Detect fetal distress and used to determine the possibility of neural tube defect (NTD) -Normal values are based on the week of gestation age. -Fetus produces maximal AFP at 12-15 week of gestation age. Serum & amniotic fluid are reported in terms of multiple of the median ( MoM) - 2 or > MoM is abnormal for both maternal serum & amniotic fluid -elevated AFP levels are followed by measurement of amniotic cholinesterase (AchE) 6. Amniotic Acetyl Cholinesterase (AChE) Activity testPurpose: A confirmatory test for high and borderline maternal AFP results to detect NTD and predict fetal distress -Acetyl Cholinesterase a nerve enzyme that diffuse into amniotic fluid like AFP but only in presence on NTD - independent of both gestational age and fetal blood contamination. Methods Used: Colorimetric Assays Electrophoretic Method Immune Chemical Assay Third Trimester Amniocentesis Sample Analysis: 1. Spectrophotometric Analysis of Amniotic Bilirubin- Purpose: Evaluate the severity of fetal anemia produced by HDN to the fetus - Bilirubin present in the fluid determines the degree of hemolysis and assess the danger that HDN to the fetus. - Amount of bilirubin can be determined from the height of the peak in the Liley Graph -The amniotic fluid is subjected to a spectrophotometric scan at steadily increasing wavelengths. - Wavelength used between 365 and 550 nm. Mental retardation and other congenital malformation.

-Liley graph plots the change in OD at 450nm versus gestational age in weeks. 2. Leukocyte Esterase Reagent Strip Test for WBC CountPurpose: detect possible infection of the mother and fetus in case of premature or prolonged rupture of the amniotic membrane. - Uses a leukocyte esterase reagent strip. - The esterase levels in amniotic fluid correlates with the number of WBC present. - WBC count > 50/ul AF is indicative of infection. 3. Thin layer chromatography (qualitative measurement of lecithin and sphingomyelin) Purpose: to determine the maturity of the fetal lung and fetal well being to prevent the occurrence of respiratory distress syndrome. Fetal Pulmonary Maturation - is marked by production of surfactants which form a film on the alveolar lining. RESPIRATORY DISTRESS SYNDROME- The resultant condition when there is deficient amount of quality of surfactant in neonates. - Most frequent complication of early delivery. Phospholipid Component of surfactants: A. Lecithin (Phosphatidyl Choline)- Major surface active phospholipids. - Accounts for alveolar stability - produced at relatively low and constants rate until the 35th week of gestation B. Phosphatidyl glycerol - Also essential in fetal lung maturation C. Sphingomyelin - No major surface active property - Used as internal STD in the reference method for L/S ratio test - Produced constantly at 26 weeks of gestation. D. Acid phospholipids E. Phosphatidylethanolamine Result in TLC = L/S ratio of > 2.0 means there is increased lecithin production, resulting in the stabilization of fetal lung alveoli. Disadvantages of L/S ratio: a. False elevated results in fluid contaminated by blood and meconium. b. The test is not specific among diabetic mothers c. The test insensitive Phosphatidyl glycerol and phosphatidyl inositol maybe used especially if fluid is contaminated with blood and meconium. AMINOSTAT FLM -an immunologic agglutination test which utilize antisera specific for phosphatidyl glycerol.

-can be performed on specimen contaminated with blood or meconium. 4. Different Biochemical techniques * Purpose: to detect presence of surface lipids and predicts fetal lung maturity status. a. Foam or shake test b. Foam Stability Index (modification of the sample shake test) c. Spectrophotometric Method (650 nm.) d. Microviscosity Measurement phospholipid decreases the microviscosity of the amniotic fluid measured using the principle of fluorescence polarization. e. Creatinine Determination determines fetal age.

C. Determine maternal from fetal blood (in case of traumatic tap) PARAMETERS 1. Kleihauer staining 2. Alkali buffer solution 3. MCV MATERNAL BLOOD Cell ghosts uncoloredempty membranes Sensitive 140 fl FETAL BLOOD Red, refractile large cells Resistant <100 fl

EXAMINATION: Tests for fetal well being and fetal lung maturity TEST 1. Bilirubin scan 2.Lecithin/ sphingomyelin ratio 3.Phosphatidyl-glycerol 4. Foam Stability index 5. Optical density (OD) 650nm 6. TDX FLM 7. Creatinine NORMAL VALUES AT TERM 0.025 mg/dL 2.0 Present > 47% > 0.250 > 70 mg/g 1.8 to 4.0 mg/dL CLINICAL SIGNIFICANCE Hemolytic disease of the newborn Fetal lung maturity Fetal lung maturity Fetal lung maturity Fetal lung maturity Fetal lung maturity METHODS Spectrophotometry plotted on Liley Curve Thin-layer chromatography Thin-layer chromatography Foam or Shake test Spectrophotometry Fluorescent polarization RIA Electrophoresis

Fetal age of = 36 weeks 8. Alpha-fetoprotein <2.5 MoM Neural tube defects 9. Acetylcholinesterase Neural tube defects Specimen should be incubated at 37Oc for no longer than 2 days if not tested immediately IDENTIFICATION OF AMNIOTIC FLUID: A. From maternal urine (as a contaminant during amniocentesis) PARAMETERS 1. Urea nitrogen 2. Creatinine MATERNAL 300 mg/dL >10 mg/dL AMNIOTIC FLUID 30 mg/dL <3.5 mg/dL

B. From cervicovaginal secretion ( in case of premature rupture of membrane) PARAMETERS 1. Nitrazine test 2. Ferning 3. Fibronectin CERVICOVAGINAL SECRETION pH 4.5 to 5.5 (+) (-) AMNIOTIC FLUID pH 7.0 to 7.5 (-) (+)