Article 274 0.1 CEC Amniotic Fluid Testing Sharon Price, Debbie Wisenor, and George H. Roberts Giving birth to a healthy baby is truly a miracle. ties were detected by American researchers using am- There are numerous things that can go wrong in a niotic fluid. The procedure became safer in the 1980’s pregnancy that can be detrimental or affect the quali- when ultrasound techniques used in World War I were ty of life of a fetus. During pregnancy, a prenatal care incorporated into obstetrics. By using the ultrasound physician offers a variety of testing that is available to image, the fetus and the fluid can be located with cer- detect fetal abnormalities. Several options in testing tainty. Procedure related miscarriage rates decreased exist for the mother and physician. Some are more in- after this technique had been incorporated (Rapp, vasive than others; therefore, the risks involved are 1999). different. Some are screening tests, while others are Amniocentesis procedures are usually performed diagnostic. Amniotic fluid testing, using fluid ex- in a physician’s office and the sample is sent to a clin- tracted by amniocentesis, is used for specific diagno- ical laboratory for testing. To extract the fluid, a sis of several genetic abnormalities and is an impor- physician uses a fine gauge needle to puncture the tant source of information to physicians about the mother’s abdomen through to the amniotic sac in re- health of the fetus. gions that are unoccupied by the fetus and withdraws To fully understand the procedure of amniocente- approximately ten mL of fluid (Burtis & Ashwood, sis an overview of the production and components of 2001). Anesthesia is generally not needed (Michigan amniotic fluid is useful. In the beginning of pregnan- State University, n.d.); however, the mother may cy, amniotic fluid is made by the amnion and later is complain of mild cramping (Himes, 1999). As men- supplied by the kidneys and lungs. The normal vol- tioned above, ultrasonography is used simultaneously ume for amniotic fluid is 500-1500 mL at full term. A to produce real-time images to help in guiding the constant temperature and cushioning are provided to needle (Burtis & Ashwood, 2001). If amniocentesis is the fetus through amniotic fluid. The composition of performed on an Rh negative woman, she should re- amniotic fluid varies greatly throughout gestation. At ceive an injection of Rh immunoglobulin to avoid its earliest, it is primarily composed of maternal possible immunization of the mother (Roberts, 1998). serum. As the fetal kidneys develop, urea, creatinine, After extraction, transferring the sample to a brown and uric acid, in addition to small amounts sodium sterile tube is necessary to prevent the breakdown of and osmolality are detected. Also, phospholipids bilirubin. In addition, if fetal lung maturity is to be along with steroid and protein hormones are found assessed, the sample must be iced during transport (Burtis & Ashwood, 2001). Karyotyping of the fetal (Burtis & Ashwood, 2001). chromosomes is facilitated by cells shed from the am- After the sample has been collected, it is sent to nion, fetal skin, bladder, gastrointestinal tract, and tra- the laboratory where it is logged in and the testing Sharon Price, Clinical cheobronchial tree around the sixteenth week of ges- procedures begin. First, the amniotic fluid is cen- Laboratory Science tation (Burtis & Ashwood, 2001; “CDC publishes,” trifuged. Subsequently, a medical technologist is able Student, The 1995). If the fetus is under stress, meconium, a stool- to assess whether or not the sample has the five per- University of like substance, may be noticed in the amniotic fluid as cent fetal cell material that is necessary to continue Louisiana at Monroe; Debbie Wisenor, MA, a green hue (Burtis & Ashwood, 2001). testing. If the cell count is adequate, most of the su- MT(ASCP), Clinical In 1882, a German physician made the first docu- pernatant is decanted and used for other testing in- Laboratory Science mented attempt at amniocentesis. Amniocentesis is cluding alpha-fetoprotein and lecithin/sphingomyelin Instructor, The simply the extraction of amniotic fluid. His motive ratio. The remainder is resuspended with growth University of was not research but therapy to a patient. He removed media and placed into three flasks. Different growth Louisiana at Monroe; George H. Roberts, amniotic fluid to relieve pressure in the amniotic sac. media may be placed in each respective flask to in- EdD, MT(AMT), For many years, physicians employed this experi- crease the chances of growth. Carbon dioxide is then Professor and mental procedure for that purpose. In the 1950’s, am- added and a 37° C incubation period of six days be- Department Head, niotic fluid became a basis for testing for hemolytic gins. If, after this time, enough growth is not avail- Clinical Laboratory disease of the newborn (HDN) due to Rh incompati- able, a food source is added until sufficient growth Science, The University of bility in pregnancy. Later, fetal lung maturity was occurs. The average time needed for cell growth is Louisiana at Monroe added to its uses. In 1967, chromosome abnormali- five to nine days, a much decreased interval from ear- 80 April 2004 • Continuing Education Topics & Issues lier days. After this, the amniocytes are ready for vantage to earlier diagnosis is that treatment may be chromosomal studies or karyotyping (Rapp, 1999). available, but if the diagnosis is delayed, the damage Karyotyping requies that cell growth be halted in the may be irreversible. SA is offered after the fifteenth metaphase stage of mitosis (Verma et al., 1998). week of gestation, while the time span for EA varies. These cells are centrifuged, the supernatant is re- Any time from nine to fifteen weeks is accepted as moved, and the cell buttons are resuspended. A hy- EA (Himes, 1999). Testing for genetic disorders potonic solution is added to lyse the cells. Following using cytogenetics with EA is equivalent to SA; how- another centrifugation and addition of fixative, the ever, the success rate of using EA to detect NTD by chromosomes are applied to glass microscope slides levels of alpha-fetoprotein and acetylcholinesterase and dried. The integrity of the suspension and chro- is not established (Kuller &Laifer, 1995). Increased mosome spread is evaluated at this point. The slides risks associated with EA have been studied. In a study are stained and dried. Chromosomes now appear to described by Himes in the Journal of Perinatal and have distinct dark, light, and intermediate gray bands Neonatal Nursing (1999), the women randomly se- that are used for judging normalcy and abnormalcy lected for the EA had a higher rate of fetal loss of of chromosomes (Rapp, 1999). 1.7% and an increased rate of amniotic fluid leakage Counting the number of chromosomes present is than did the SA participants. A very significant in- the first action in analysis (Rapp, 1999, 1999). Kary- crease of talipes equinovarus (club foot) was noted in otype images are created using chromosome kary- the EA group (Sundberg et al., 1997). In addition, or- otyping analysis. Usually, chromosomes are orga- thopedic and respiratory problems have been shown nized from longest to shortest. By arranging the to occur after birth in EA patients. These problems chromosomes, anomalies are more readily seen may be linked to the time period during which the (Xiong, Wu, & Castlemen, n.d.). Twenty cells must fluid is collected which is during limb and lung de- be counted. If there is an abnormality, thirty cells are velopment (Himes, 1999). An additional study by analyzed. At thirty cells, the diagnosis is confirmed. Kuller & Laifer discussed in American Family Physi- Several other investigative circumstances may arise cian (1995) supported these results. In addition to am- and a protocol exists for each one. Finally, abnormal niotic fluid leakage, vaginal bleeding was more slides are always inspected by additional personnel prominent in patients undergoing EA. This study to confirm the diagnosis (Rapp, 1999). found that there was a higher rate of pregnancy loss in In addition to chromosome studies, other compo- EA during the thirty days immediately following the nents of the amniotic fluid are tested as well to deter- procedure, but after this period the outcome of preg- mine the health of the fetus. Determining fetal lung nancy was essentially equivalent among the two maturity is important in detecting Respiratory Dis- groups (“Complications,” 1996). tress Syndrome (RDS) and aids the physician in mak- Why would a physician risk these adverse effects? ing the decision to facilitate labor. To do this the Amniotic fluid testing is used to inform the family lecithin/spingomyelin (L:S) ratio is used. A ratio of and their physician about the health of the fetus. The 2:1 or greater indicates that the fetal lungs should be main reason mothers undergo amniocentesis is to di- mature enough to function outside the uterine envi- agnose or rule out Down syndrome. In addition to ronment. The most widely used quantitative tech- this, other genetic anomalies such as trisomy 13 and nique to determine fetal lung maturity is fluorescence trisomy 18 are detectable. All three of these disorders polarization. Bilirubin in the amniotic fluid may also have increased rates of occurrence with increased ma- be measured to indicate hemolytic disease of the ternal age (Mennuti, 1996). Neural tube defects can newborn (HDN). The most common occurrence of also be detected using amniotic fluid (Stevenson et HDN is with Rh negative mothers and Rh positive ba- al., 2000). High levels of AFP and acetyl- bies. Bilirubin is measured to determine the severity cholinesterase are detected in the presence of a NTD of the disease. It is measured by using absorption (Kuller & Laifer, 1995). Also, if an intra-amniotic in- spectrophotometry.