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Amniotic Fluid

Purpose of examination
Cytogenetic analysis A product of fetal metabolism Provide information about the metabolic processes taking place during fetal maturation *the danger to the fetus must be measured against the ability of the fetus to survive an early delivery

Physiology

function
Amnion-contains the amniotic fluid; a membranous sac that surrounds the fetus A protective cushion for the fetus Allow fetal movement Stabilize the temperature to protect the fetus from extreme temperature changes

function
Permit proper lung development Medium for exchange of water and chemicals between fetus and maternal circulation

volume

It is regulated by a balance between the production of fetal urine and lung fluid and the absorption from fetal lung swallowing and intramembranous flow

Intramembranous flow

Is the absorption of amniotic fluid water and solutes into the fetal vascular system

volume
Amount of amniotic fluid increases during pregnancy Peak of about 1L during the 3rd trimester Gradually decreases prior to delivery First trimester: 35 mL is derived from the maternal circulation

volume
Half to latter third of pregnancy, the fetus secretes a volume of lung liquid necessary to expand the lungs with growth Each fetal breathing movement secretes lung surfactants that serve as an index of fetal lung maturity

volume
After the first trimester, fetal urine is the major contributor to the amniotic fluid volume During fetal urine production, fetal swallowing of the amniotic fluid begins

Polyhydramnios
Failure of the fetus to begin swallowing results in excessive accumulation of amniotic fluid An indication of fetal distress Often associated with neural disorders

polyhydramnios
Secondarily associated with: Fetal structural anomalies Cardiac arrhythmias Congenital infections Chromosomal abnormalities

oligohydramnios
Decreased amniotic fluid Causes: Increased fetal swallowing Urinary tract deformities Membrane leakage Umbilical cord compression resulting in decelerated heart rate and fetal death

Chemical composition
Placenta-the ultimate source of amniotic fluid Amniotic fluid has a composition similar to maternal plasma and a small amount of sloughed cells from the skin,digestive system,and urinary tract (provides the basis for cytogenetic analysis

Chemical composition
Biochemical substances produced by the fetus: Bilirubin Lipids Enzymes Electrolytes Nitrogenous compounds Proteins

Chemical composition
Fetal respiratory tract Fetal urine Amniotic membrane Umbilical cord

Fetal urine production


Creatinine,urea,and uric acid increase Glucose and protein decrease Electrolytes,enzymes,hormones,and metabolic end products vary

Amniotic fluid creatinine


Prior to 36 weeks gestation,AF creatinine ranges between 1.5 and 2.0 mg/dL After 36 weeks, it rises above 2.0 mg/dL thereby providing a means of determining fetal age as greater than 36 weeks

Differentiating maternal urine from amniotic fluid


To determine possible premature membrane rupture or accidental puncture of the maternal membrane during specimen collection Creatinine and urea are much lower in amniotic fluid than in urine

Amniotic fluid Creatinine is </= 3.5 mg/dL Urea is </= 30 mg/dL Urine Creatinine is >/= 10 mg/dL Urea is >/= 300 mg/dL *fern test

Specimen Collection

Indications for amniocentesis


Abnormal results for the following: Maternal serum AFP test Triple screening test(AFP,hCG,UE3) Quadruple screening test (AFP,hCG,UE3,inhibin A) Abnormal results in ultrasound Intrauterine growth retardation

Fetal epithelial cells


Fluorescence in situ hybridization(FISH) Fluorescent mapping spectral karyotyping(SKY) DNA testing

Biochemical substances
Fluorescence polarization Thin-layer chromatography

Indications for amniocentesis


15 to18 weeks of gestation for the ff conditions: Mothers age of 35 or more at delivery Family history of chromosome abnormalities such as trisomy 21 Parents carry an abnormal chromosome rearrangement Earlier pregnancy or child with birth defects

Parent is a carrier of a metabolic disorder History of genetic diseases ex. CF Elevated maternal serum alpha fetoprotein Abnormal triple marker screening test Previous child with a neural tube disorder such as spina bifida,or ventral wall defects (gastroschisis) Three or more miscarriages

Amniocentesis indicated at 20 to 42 weeks to evaluate: Fetal lung maturity Fetal distress Hemolytic disease of the newborn caused by Rh blood type incompatibility infection

collection
Amniocentesis-collection of amniotic fluid by needle aspiration into the amniotic sac Transabdominal amniocentesis Safer when performed after the 14th week of gestation 16th week-chromosomal analysis 3rd trimester-tests for fetal distress and maturity

collection
Maximum of 30 mL is collected in sterile syringes First 2 to 3mL are discarded due to contamination *for bilirubin analysis due to HDNprotect from light; use amber-colored tubes or black colored covers

Specimen handling and processing


Fetal lung maturity tests(FLM) tests Place in ice for delivery and refrigerate up to 72 hours prior to testing or kept frozen and tested within 72 hours Frozen specimens should be mixed after thawing Cytogenetic analysis-maintain at 37C body temperature or room temperature prior to analysis

Specimen handling and processing


Chemical testing Fluids separated by centrifugation or filtration Filtration-recommended for FLM methods to prevent loss of the phospholipids

Color and Appearance


Colorless-normal Slight to moderate turbidity due to cellular debris Blood streaked fluid Traumatic tap Abdominal trauma Intra-amniotic hemorrhage Kleihauer-Betke test-determines fetal Hgb

Color and appearance


Yellow color bilirubin indicative of rbc destruction from HDN Dark green color meconium newborns first bowel movement fetal intestinal secretions Red-brown fluid associated with fetal death

Tests for Fetal Distress

Hemolytic disease of the newborn


Bilirubin Spectrophotometric analysis OD is measured between 365 nm and 550 nm

Neural Tube Defects


Increased levels of alpha-fetoprotein in both the maternal circulation and the amniotic fluid AFP-major protein produced by the fetal liver during early gestation (prior to 18 weeks);maximal at 12-15 weeks AOG Anencephaly Spina bifida

Indications for NTDs


Maternal serum levels are elevated Family history

AChE
Amniotic acetylcholinesterase More specific for neural tube disorders

Tests for Fetal Maturity

Fetal lung maturity


Respiratory Distress Syndrome(RDS) Is the most frequent complication of early delivery Lack of lung surfactant- a substance that normally appears in mature lungs and allows the alveoli(air sacs of the lung) to remain open throughout the normal cycle of inhalation and exhalation

Lung surfactant

Keeps the alveoli from collapsing by decreasing surface tension and allows them to inflate with air more easily

Lecithin-Sphingomyelin Ratio
L/S ratio Lecithin-primary component of the surfactants (phospholipids,neutral lipids, and proteins) that make up the alveolar lining and account for alveolar stability

lecithin

Produced at a constant rate until 35th week AOG then increases

sphingomyelin
Is a lipid produced at a constant rate after 26 weeks AOG Serves as a control on which to base the rise in lecithin

L/S ratio
Less than 1.6 prior to 35 weeks AOG Rises to 2.0 after 35 weeks AOG Ratio of 2.0 means safe to do a preterm delivery Falsely increased with blood or meconium contamination TLC-quantitative measurement

Amniostat-FLM
Phosphatidyl glycerol-lung surface lipid essential for adequate lung maturity Delayed production in maternal diabetes Respiratory distress=L/S ratio of 2.0 TLC Agglutination test

Foam Stability
foam or shake test mechanical screening test used to determine lung surfactants Amniotic fluid is mixed with 95% ethanol, shaken for 15 seconds,and stand for 15 minutes,then observed for presence of bubbles around the outside edge

Microviscosity: Fluorescence Polarization Assay


The presence of phospholipids decreases the microviscosity of amniotic fluid Measures the polarization of a fluorescent dye that combines with both surfactants and albumin

principle

Dye bound to surfactant has a longer fluorescence lifetime and exhibits low polarization whereas dye bound to albumin has a decreased fluorescence lifetime and has a high polarization

FLM=/<55 mg surfactant per gram albumin Immature results=/< 39 mg/g Test requires at least 1.0 mL of amniotic fluid

Lamellar Bodies and Optical Density


Lamellar bodies-are lamellated phospholipids that represent a storage form of surfactant Type II pneumocytes 20 weeks AOG Principle: the presence of lamellar bodies increases the OD of the amniotic fluid

Tests
Lamellar body counts (LBCs) with use of hematology analyzers(similar to platelets) Resistance-pulse counting(Beckman Coulter) 1.7 to 7.3 fL FLM=/> 32,000/uL ADVIA 120 hema system(Siemens) FLM=/> 35,400 particles/uL Sysmex XE-2100-impedance Cell-dyn 3500 (Abbott Lab)-optical scatter

Lamellar body count

ADVIA 120 hematology system

Sysmex XE-2100

Cell-dyn 3500 (Abbott Lab)

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