Effect of blood contamination on delta 450 bilirubin measurement: an in vitro comparison of two corrective methods.

Abstract

Amniotic fluid bilirubin peaks (1.6–1.8 mg/L) at ∼19–22 weeks of gestation (1). An increase in amniotic fluid bilirubin concentration after 25 weeks indicates extensive hemolysis of fetal blood and potential erythroblastosis fetalis in which amniotic fluid bilirubin can reach ∼10 mg/L (2)(3). Bilirubin in amniotic fluid can be measured by direct spectrophotometry, most commonly by the “delta 450 bilirubin” method (4). Contaminating pigments, such as hemoglobin, can interfere in the method. Chloroform extraction has been used to extract bilirubin, leaving water-soluble pigments in the aqueous layer. The delta 450 bilirubin is then measured in the chloroform fraction and interpreted by use of the Liley plot (5)(6). A delta 410 correction formula also exists that theoretically accounts for hemoglobin interference. The delta 410 absorbance is measured and used to correct the delta 450 bilirubin (4)(6)(7). Our objective was to examine the effect of hemolyzed-whole-blood contamination on the delta 450 bilirubin measurement and to compare corrective methods. After receiving Institutional Review Board approval, we pooled amniotic fluid specimens collected for physician-ordered fetal lung maturity assessment (stored at −20 °C). Visibly bloody or meconium-contaminated samples were not used. The pool was centrifuged at 1100 g for 5 min, and the supernatant was filtered through a 22 μm AP 20 prefilter (Millipore Corp.) and a 1.2 μm RA filter (Millipore). Bilirubin was undetectable in the pool by delta 450 measurement. We prepared a 600 mg/L stock solution of unconjugated bilirubin (ICN Biomedicals Inc.) by dissolving 6.0 mg of bilirubin in 0.1 mL of dimethyl sulfoxide (Sigma Chemical Co.) and 0.2 mL of sodium carbonate (100 mmol/L). The solution was vortex-mixed until the bilirubin was dissolved. We then added 9.5 mL of pooled amniotic fluid, followed by 0.2 mL of HCl (100 mmol/L) to …