Rapid spectrophotometric measurements of total bilirubin in intact and hemolyzed neonatal blood: a feasibility study.

Abstract

Conventional methods for measuring the total bilirubin concentration in blood require the use of serum or plasma, but physically separating red blood cells from plasma by centrifugation is a time-consuming and potentially dangerous process that does not lend itself to rapid, near-patient testing. Therefore, we have sought to determine whether spectrophotometric measurements of total bilirubin concentration are feasible in unaltered whole blood. We modified an Hb-Quick hemoglobinometer (Avox Systems, Inc., San Antonio, TX), a relatively new, portable, battery-powered instrument that uses disposable cuvettes and a reagentless system to measure total hemoglobin in nonhemolyzed, whole blood. The prototype consisted of an Hb-Quick equipped with light-emitting diodes with emissions at five different wavelengths to measure total bilirubin and total hemoglobin. Using blood samples from neonates with suspected hyperbilirubinemia, we made measurements on plasma from centrifuged samples and on hemolyzed and nonhemolyzed aliquots of the same blood samples. In the first series of experiments, we compared the Unistat bilirubinometer's readings with the prototype's measurements of bilirubin in plasma. There was a close linear correlation between the prototype's measurements and those of the reference instrument (slope=1.01, r(2)=0.991). Subsequently, we used the prototype's measurements on plasma as the reference method and compared them with readings on hemolyzed and nonhemolyzed aliquots of each sample. Readings on hemolyzed and nonhemolyzed aliquots were significantly correlated with the measurements in plasma, but the regression lines did not have a slope of 1. However, when the measurements on hemolyzed and nonhemolyzed blood were scaled appropriately to compensate for the fact that red cells "dilute" the bilirubin in plasma, the correlation coefficients improved, and there was then a 1:1 relationship between the measurements in whole blood and plasma. These preliminary findings demonstrate the feasibility of developing a portable instrument to measure total bilirubin in unaltered whole blood. The advantages of this method are speed, elimination of centrifugation or other sample preparation, and instrument portability. The disadvantage is that the concentration units are unconventional, i.e., milligrams of bilirubin per volume of whole blood. However, the instrument can be programmed to display the total bilirubin concentration in traditional units, e.g., milligrams of bilirubin per volume of plasma.