Urinalysis and other Body Fluids - Amniotic Fluid Analysis.pdf

ANALYSIS OF URINE AND OTHER BODY FLUIDS • AMNIOTIC FLUID ANALYSIS • OUTLINE • • Introduction Amniotic Fluid o Physiology o Specimen Collection o Specimen Handling and Processing o Color and Appearance o Tests for Fetal Distress o Test for Fetal Lung Maturity • • • • INTRODUCTION • Amniotic fluid refers to the liquid medium that bathes the fetus during the gestational period. This fluid protects the fetus and allows for free movement of the fetus. The amniotic fluid also plays an important role in may biochemical processes involving the fetus. The primary reasons for testing of amniotic fluid are for: antenatal diagnosis of genetic and congenital disorders, evaluation of fetal lung maturity, and monitoring the degree of fetal distress. • AMNIOTIC FLUID • • • • Amniotic fluid is a product of fetal metabolism. The constituents in the fluid provide information about the metabolic processes that taking place and the progress of fetal maturation. Amniotic fluid is present in the amnion, a membranous sac that surrounds the fetus. Functions o It provides a protective cushion for the fetus. o It allows movement for the fetus. o Exchanges of water and chemicals also take place between the fluid, fetus, and maternal circulation. PHYSIOLOGY • • • • • • The amount of amniotic fluid increases throughout pregnancy, reaching a peak of approximately 1L during the third trimester, and then gradually decreases prior to delivery. During the first trimester, the approximately 35 mL of amniotic fluid id derived primarily from the maternal circulation. After the first trimester, fetal urine is the major contributor to the amniotic fluid volume. When fetal urine production occurs, fetal swallowing of the amniotic fluid begins and regulates the increase in the fluid from the fetal urine. Failure of the fetus to swallow results in excessive accumulation of amniotic fluid (hydramnios) and is indication of fetal distress, often associated with neural tube disorders. Increased fetal swallowing, urinary tract deformities, and membrane leakage are possible causes of decreased amniotic fluid (oligohydramnios). Chemical composition of the amniotic fluid changes when fetal urine production begins. Creatinine, Urea and Uric Acid concentrations increases, whereas glucose and protein concentrations decreases. Amniotic fluid creatinine has been used to determine fetal age. Prior 36 weeks gestation yields between 1.5 and 2.0 mg/dL Creatinine level. Differentiation of amniotic fluid and maternal urine may be necessary to determine possible premature membrane rupture or accidental puncture of the maternal bladder during collection. Chemical analysis of creatinine, urea, glucose, and protein will aid in the differentiation Tests for Fetal Well-Being and Maturity Test Normal Values Significance at Term16 Bilirubin scan ∆A450 >0.025 HDN Alpha-fetoprotein <2.0 MoM Neural Tube disorders Lecithinsphingomyelin >2.0 Fetal Lung maturity ratio Amniostat-fetal lung Positive Fetal lung maturity maturity / phosphatidyl glycerol Foam Stability Index >47 Fetal Lung maturity Microviscosity >70 mg/g Fetal Lung maturity Optical density 650 >0.150 Fetal Lung maturity nm Lamellar body count >32 000/μL Fetal Lung maturity SPECIMEN COLLECTION • • • • • • Amniotic Fluid is collected through needle aspiration into the amniotic sac, a procedure called amniocentesis. Vaginal amniocentesis may also be performed but carries a greater risk of infection. Amniocentesis is a safe procedure performed after the 14th week of gestation A maximum of 30 mL of amniotic fluid is collected in sterile syringes. The first 2 or 3 mL collected can be contaminated by maternal blood , tissue fluid, and cells and are discarded. Specimen used for bilirubin test in cases of HDN must be protected from light at all times and can be accomplished by placing the specimens in amber-colored tubes. SPECIMEN HANDLING AND PROCESSING • • • • • Tests for Fetal Lung Maturity should be placed in ice for delivery to the laboratory and refrigerated prior to testing. Specimens for cytogenic studies are maintained at room temperature or body temperature (37°C incubation) prior to analysis to prolong the life of the cells nede for analysis. All fluid for chemical testing should be separated from cellular elements and debris as soon as possible to prevent distortion of chemical constituents by cellular metabolism or disintegration. This can be performed using centrifugation or filtration. Low-speed centrifugation (500-1000g) for no longer than 5 minutes is required for FLM testing. At higher speed some phospholipids (lung surfactants) may be lost in the sediment. o COLOR AND APPEARANCE Color Colorless Blood-streaked Yellow Dark green Dark red-brown Significance Normal Traumatic tap, abdominal trauma, intraamniotic hemorrhage HDN (Bilirubin) Meconium Fetal death o Elevated amniotic fluid AFP levels are followed by measurement of amniotic acetylcholinesterase (AChE). The test is more specific for neural tube disorders than AFP, provided it is not performed on a bloody specimen, because blood contains AChE. TESTS FOR FETAL DISTRESS • Hemolytic Disease of the Newborn o Oldest routinely performed laboratory test on amniotic fluid. o When antibodies present in the maternal circulation cross the placenta, the destruction of fetal red blood cells results in the appearance of the red blood cell degradation product, bilirubin, in the amniotic fluid. o The measurement of amniotic fluid bilirubin is performed by spectrophotometric analysis. o Values falling in zone I indicate no more than a mildly affected fetus; in zone II require careful monitoring, whereas in zone III suggests a severely affected fetus. o Markedly decreased values will be obtained with as little as 30 minutes of exposure to light. o Care must be taken to ensure that contamination of the fluid by cells, hemoglobin, meconium, or other debris does not interfere with the spectrophotometric analysis. o This interference can be removed by extraction with chloroform if necessary. TESTS FOR FETAL LUNG MATURITY • • • Neural Tube Defects o Increased levels of alpha-fetoprotein (AFP) in both the maternal circulation and the amniotic fluid can be indicative of fetal neural tube defects, such as anencephaly and spina bifida. o AFP is the major protein produced by the fetal liver during early gestation. o It is found in the maternal serum due to the combined fetal-maternal circulations and in the amniotic fluid from diffusion and excretion of fetal urine. o Both serum and amniotic fluid AFP levels are reported in terms of multiples of the median (MoM). o Normal values are based on the week of gestational age, as the fetus produces maximal AFP between 12 and 15 weeks’ gestation, after which levels in amniotic fluid then begin to decline. o Testing for AFP levels had been automated by the Access Immunoassay System. • Lecithin-Sphingomyelin Ratio o Lecithin is the primary component of the surfactants (phospholipids, neutral lipids, and proteins) that make up the alveolar lining and account for alveolar stability. o Lecithin is produced at a relatively low and constant rate until the 35th week of gestation. o Both lecithin and sphingomyelin appear in the amniotic fluid in amounts proportional to their concentrations in the fetus. o Falsely elevated results are encountered in fluid contaminated with blood or meconium because both these substances contain lecithin and sphingomyelin. o Quantitative measurement of lecithin and sphingomyelin is performed using thin-layer chromatography. o Many laboratories have replaced the L/S ratio with the more cost-effective phosphatidyl glycerol immunoassays, fluoresence polarization,a nd lamellar body density procedures. Amniostat-FLM o The presence of phosphatidyl glycerol, is also essential for adequate lung maturity. o Development of an immunologic agglutination test for phosphatidyl glycerol has provided a more rapid method for assessment of fetal maturity that does not require a laboratory to be equipped to perform thinlayer chromatography. o The Amniostat-FLM uses antisera specific for phosphatidyl glycerol and is not affected by specimen contamination with blood or meconium. Foam Stability o “Foam” or “shake” test, is used to measure individual lung-surface lipid concentrations. o Because it can be performed along bedside or in the laboratory, the test is still in use. o Amniotic fluid is mixed with 95% ethanol, shaken for 15 seconds, and then allowed to sit undisturbed for 15 minutes. Presence of continuous line of bubbles around the ouside edge is then observed. o A value of 47 or higher indicates FLM o The test cannot be used with contaminated amniotic fluid because blood and meconium will also reduce surface tension. • • • Microviscosity o Presence of phospholipids decreases the microviscosity of amniotic fluid. o Change in microviscosity can be measured by the Abbott TDx analyzer using the principle of fluoresence polarization. o The instrument measures the polarization of fluorescent dye that combines with both surfactants and albumin. o Fluid should be filtered rather than centrifuged prior to examination to prevent sedimentation of the lipids, and use of contaminated fluid is not recommended. Lamellar Bodies and Optical Density o The number of lamellar bodies present in the amniotic fluid correlates either the amount of phospholipid present in the fetal lungs. o The specimen is examined at 650 nm, which rules out interference from hemoglobin but not from meconium. o Lamellar bodies can be counted using resistancepulse counting such as employed by Coulter cellcounting instruments. o Lamellar bodies can be counted using the platelet channel. o This technique is easily performed; however, samples must be free of particle contamination such as meconium and blood. o A count of 32 000 or more particles per microliter represents adequate FLM.