CLINICAL APPLICATION OF A NEW CHEMICAL METHOD FOR THE ESTIMATION OF BILIRUBIN IN LIQUOR AMNII

IN recent years the estimation of bilirubin in the liquor amnii has been increasingly used as a guide to the management of haemolytic disease due to rhesus iso-immunization. Two methods have been used; an assessment of the spectral absorption curve at 450 mp (Walker, 1957; Liley, 1961 ; Alvry, 1964), and a chemical estimation based upon the diazo reaction (Lathe and Ruthven, 1958; Stewart and Taylor, 1964). The disadvantages of the former method are that dissolved oxyhaemoglobin produces a number of peaks which interfere with absorption at the critical wavelength for bilirubin, and also that it is not possible to obtain a reading in terms of the actual concentration of bilirubin in the amniotic fluid. In the chemical method, unless specific modifications are made to the diazo reaction the low concentrations of bilirubin found in the liquor require that readings are made at the limits of sensitivity of the method. In this investigation we have made use of the extreme solubility of unconjugated bilirubin in chloroform (Pennington and Hall, 1966). The bilirubin is extracted from the liquor by shaking with chloroform; other compounds which might interfere with the estimation are not extracted. The resulting dilute solution is then concentrated by evaporation; and by reference to a standard curve the bilirubin may be estimated quantitatively. This method is extremely quick and simple to use and a result can be obtained in under ten minutes.