Determination of Bilirubin in Amniotic Fluid: A Comparison of Some Current Methods

Abstract

Three methods for the estimation of bilirubin in amniotic fluid are compared. The within-batch precision and recovery of bilirubin by each method has been studied, and the effects of the presence of haemoglobin, methaemoglobin, and methaemalbumin in varying concentrations have been investigated. Measurements on amniotic fluid from patients with rhesus isioimmunization have also been compared and regression equations calculated. The results of the three methods agree reasonably well so long as no haemoglobin degradation products are present. It is suggested that in the presence of these products a method involving organic solvent extraction of bilirubin gives the most accurate result.

Studies of the Extraction of Bilirubin From Human Amniotic Fluid

“…1,2 Because interference in measuring bilirubin absorbance may occur when blood contaminates the amniotic fluid, extraction has been advocated to recover the bilirubin free from hemoglobin. [4][5][6] The method of Hochberg and coworkers 4 using chloroform has been the most widely used in this regard.…”

“…Because amniocentesis often results in contamination of the amniotic fluid specimen with maternal and/or fetal blood such that the Soret band of hemoglobin (absorbance at 410 nm) interferes with the measurement of bilirubin absorbance, chloroform extraction of the fluid was advocated by Hochberg and coworkers 4 in 1976. Subsequently, organic solvent extraction has been recommended as providing the most accurate result for measurement of bilirubin absorbance in the presence of hemoglobin, 5 and chloroform extraction methods have been held to accurately predict fetal status. 6 Recently, however, Foster and associates 7 suggested that chloroform extraction of bilirubin from amniotic fluid decreases the measured bilirubin absorbance by more than 60% owing to incomplete recovery of bilirubin by chloroform.…”