AMNIOTIC FLUID

Found in the membranous sac that surrounds the fetus providing a cushion for protection Formed from metabolism of fetal cells, transfer of water across the placental membrane & in the 3rd trimester by fetal urine Obtained by Amniocentesis Normal volume: 0.5-1.5 L Increased Amniotic fluid volume: Acute hydramnios, Chronic hydramnios Decreased Amniotic fluid volume: Oligohydramnios

Optical density: Examined at 650 nm Normal value: >0.250 Amniotic particle counting (Lamellar bodies) Fetal maturity:> 32,000 particles /microliter AMNIOTIC FLUID Alkaline <30 mg/dl <3.5 mg/dl (+) (+) (+) MATERNAL URINE Acidic >300mg/dl >10 mg/dl (-) (-) (-)

TEST pH UREA CREATININE PROTEIN GLUCOSE FERN TEST

Indications: Early (14-16 weeks): Diagnosis of genetic diseases Mid Trimester: Bile pigments level for HDN Neural tube defects 3rd Trimester: Renal maturity Bile pigments level for HDN

FETAL RED CELLS & MATERNAL RED CELLS: Kleihauer-Betke Stain Fetal RBC: Red, refractile, large cells, resistant to alkali Maternal RBC: Uncolored ghost cells ore dull red, poorly stained, sensitive to alkali Immunofluorescence staining of Hgb F Flow cytometry Electrophoresis or Isofocusing of Hgb F Electronic counting

Fetal Distress Analysis: Measurement of bilirubin in the amniotic fluid by spectrophotometric analysis & plotted in the Lilley curve Normal: Optical density will rise at 365mu & will decrease linearly (+) Bilirubin: Optical density will be seen at 450 mu Elevated in Hemolytic Disease of the Newborn (HDN) / Erythroblastosis fetalis Leukocyte esterase reagent strip: Good indicator for the presence of infection WBC Count: >50/uL is Positive for infection Alpha Feto Protein (AFP): Determine neural tube disorders Major protein produced by the fetal liver & found in the maternal serum Neural tube defects: Increased Maternal serum AFP & Very high amniotic fluid AFP Acetylcholinesterase Test: Confirmatory test for neural tube disorders

FEMALE FERTILITY STUDIES Study of ovulatory & menstrual cycle Follicular / Proliferative phase hyperplasia of uterus/endometrium Ovulatory phase oocyte is already matured Increased body temperature Increased cervical mucus Weight gain Pelvic pain Irritability Luteal/Secretory phase ovary luteolysis Menstruation : regular, periodic, physiologic extrusion of blood, mucus & cellular debris from the uterus Menarche: 1st menstruation Menopause: cessation of menstruation

Fetal Maturity Analysis Lecithin/Sphingomyelin Ratio (L/S ratio): Reference method to measure fetal lung maturity Lecithin: Primary component of the phospholipids that make up the majority of the alveolar lining & account for alveolar stability Sphingomyelin: Lipid that is produced at a constant rate after about the 26th weeks of gestation serving as a control on which to base the rise in Lecithin. 2.0 L/S Ratio: Safe procedure for pre-term delivery Analytical errors: Overcentrifugation, Blood contamination Imprecision of TLC, Meconium Phosphatidylglycerol & Phosphatidylinositol: Essential for adequate fetal lung maturity (Amniostat) Foam test: Mechanical screening test to measure the individual lung surface lipid concentration Foam stability index: Good correlation with L/S Ratio Optical Density: 650nm :>/0.250 (Normal for fetal lung maturity) Depend on longevity of bubbles (ethanol-antifoaming agent) Simple shake test Manual foam stability index TDx-FLM: Utilize fluorescence polarization between lipids & albumin for fetal lung maturity Measurement of microviscosity >70 mg/g Creatinine: Detects fetal age >2.0 mg/dL: indicates approximately 36 weeks Renal maturity

FERTILIZATION Ovum must be fertilized within 1-2 days If the ovum is not fertilized, luteolysis occurs Ampulla of fallopian tube: fertilization takes place Within 6-8 days Zygote will migrate to the upper portion of the uterus HCG works when progesterone is decreased Estrogen Increased in Follicular phase FSH & Luteinizing hormone Increased after ovulation Progesterone Increased in luteal phase Decreased progesterone = Increased HCG

HUMAN CHORIONIC GONADOTROPIN (HCG) Detectable 10-14 days after conception Dimeric Glycoprotein Secreted by syncytiotrophoblast Properties: Polypeptide chains Alpha subunits: 14,000 Daltons Beta subunits: 22,000, specific

Peak levels 60-80 days after the last menses then decreases concentration: 100,000mIU/mL or 80,000 ng/mL

HCG & other diseases 1. ECTOPIC PREGNANCY 2. ABORTIONS 1.5-3.5 of HCG titer absence of doubling HCG titer <1-31 days in the blood Spontaneous: non-detectable within 9-35 days (median=49days) Induced: non-detectable within 1660 days (median=30 days) Presence of more immature cells=Increased HCG titer Elevated HCG even after pregnancy H-mole Invasive mole(myoma) Choriocarcinoma (Increased beta sub-unit) Germinoma Testicular tumors Seminoma Teratoma Gastric, pancreatic, cervical, ovarian carcinoma Poor differentiation from trophoblastic tumors

IMPORTANCE OF PREGNANCY TESTS Confirm normal pregnancy. Evaluate abnormal pregnancy. Determine if there has been complete or incomplete abortion. Differentiate Gestational Trophoblastic disease (myoma/H-mole) & other pelvic disease.

3.GESTATIONAL TROPHOBLASTIC DISEASE

4. OVARIAN GERM CELL TUMORS 5. TESTICULAR TUMORS 6. NONTROPHOBLASTIC TUMORS

IMMUNOLOGIC ASSAYS Hemagglutination Inhibition (HAI) RBC covered/coated with HCG incubated with antiHCG serum + urine 150-4,000mIU/mL (sensitivity) Latex Agglutination Inhibition (LAI) HCG coated particle of latex + urine 500-350,000 mIU/mL (+)Pregnancy: no agglutination or milky suspension Direct Latex Agglutination Antibody to HCG coated on RBC or on latex particles (+)Pregnancy: presence of agglutination Enzyme Immunoassay; Complement fixation; Sol Particle Immunoassay RADIOIMMUNOASSAY Competitive binding assay Both HCG & radio-labeled HCG compete for binding with anti-HCG Interpretation: Increased HCG=Decreased RIA=(+)Pregnancy Decreased HCG=Increased RIA = (-)Pregnancy

Other immunologic assays RADIO RECEPTOR ASSAY Uses tissues as binding site IMMUNOMETRIC ASSAY Uses radio labeled antibody IRMA: ImmunoRadioMetric Assay IEMA: ImmunoEnzymatic Assay ELISA: Enzyme Linked ImmunoSorbent Assay