One of the oldest uses of amniotic fluid analysis is in evaluating suspected hemolytic disease of the newborn, in which the mother builds antibodies against fetal red blood cell antigens (isoimmunization). The result is hemolysis of fetal erythrocytes with release of bilirubin into the amniotic fluid. The most common causes are ABO and Rh incompatibilities (e.g., an Rh-negative mother carrying an Rh-positive fetus), although other red cell antibodies may also be involved. Maternal IgG antibodies may cross the placenta to react with fetal red blood cells as early as the 16th week of pregnancy. As fetal red blood cells are broken down, bilirubin is released and can be detected in the amniotic fluid.¹¹

Normally, the bilirubin level in amniotic fluid is highest between the 16th and 30th weeks of gestation. Much of this bilirubin is in the unconjugated form and can be excreted by the placenta. As the fetal liver matures, it begins to conjugate the bilirubin; this can occur as early as 28 weeks of gestation. The conjugated bilirubin is not, however, cleared by the placenta; instead, it is excreted by the fetal biliary tract and absorbed by the intestine. After the 30th week of gestation, the bilirubin level in amniotic fluid normally decreases as pregnancy progresses. This is partly because of dilution of any bilirubin present by the normal increase in amniotic fluid volume. At term, bilirubin is nearly absent from amniotic fluid.¹²

In hemolytic disease of the newborn, fetal red cell destruction leads to excessive bilirubin levels, which overwhelm both placental and fetal liver mechanisms for its clearance. Bilirubin levels in amniotic fluid continue to rise throughout the pregnancy and consist primarily of unconjugated bilirubin.¹³ The amount of bilirubin present in the amniotic fluid indicates the degree of fetal red hemolysis and, indirectly, the degree of fetal anemia.

When hemolytic disease of the newborn is suspected or if maternal IgG levels are elevated, or both, serial amniocenteses for bilirubin determinations are performed beginning at approximately the 26th week of pregnancy. Bilirubin measurement in amniotic fluid is performed by spectrophotometric analysis, with the optical density (OD) of the fluid measured at wavelength intervals between 365 and 550 mm. When excessive bilirubin is present, a rise in OD at 450 mm, the wavelength of maximum bilirubin absorption, is seen.¹⁴ The results of spectrophotometric analysis can be compared with the Liley graph (Fig. 10-2) to predict fetal outcome or to plan medical management of the problem.

Substances other than bilirubin may cause abnormal spectrophotometric results. Maternal hemoglobin from a traumatic amniocentesis, methemalbumin, and meconium in amniotic fluid may cause false elevations, as will fetal acidosis. Fetal hemoglobin can be differentiated from maternal hemoglobin by staining and cytologic techniques. The presence of methemalbumin indicates marked hemolysis and impending fetal demise.¹⁵ Falsely decreased bilirubin levels can occur if the amniotic fluid sample is exposed to light or if excessive amniotic fluid volume causes dilution. Other disorders that can cause elevated amniotic fluid bilirubin levels include anencephaly and intestinal obstruction.¹⁶