Evaluation of the Efficacy of Chloroform Extraction of Amniotic Fluid Bilirubin

Abstract

The presence of increased concentrations of bilirubin in amniotic fluid is an indicator of fetal hemolysis (1)(2). In the Δ A 450 (or ΔOD450) method, the absorbance at 450 nm is used as a surrogate for concentration of amniotic bilirubin. Chloroform extraction of bilirubin from amniotic fluid samples can be used to eliminate spectral interference from hemoglobin, which absorbs at a maximum at 410 nm. However, clinical interpretation of the Δ A 450 often relies on studies in which chloroform extraction was not used (3)(4). We studied the extraction of bilirubin from 37 amniotic fluid samples submitted for bilirubin scanning. All studies were in accordance with the guidelines approved by the Institutional Review Board of the University of Utah. Frozen (−70 °C), light-protected samples were thawed and centrifuged at 900 g for 5 min at 2–6 °C. Absorbances of samples were scanned (Beckman DU-800) from 350 to 550 nm in 1-mL air-blanked quartz cuvettes. The use of water or chloroform blanks increased Δ A 450 <0.01 in both native and chloroform-extracted samples (n = 4). The Δ A 450 was determined as the difference between the absorbance at 450 nm and a logarithmic …