Multivariate spectrophotometric assay for cerebrospinal fluid bilirubin

Cerebrospinal fluid (CSF) contains various pigments that may contribute to discoloration termed xanthochromia. These pigments include bilirubin, hemoglobin, and proteins. Detection of bilirubin in CSF is an indication of in vivo breakdown of hemoglobin, and has been used as a diagnostic tool for detecting subarachnoid hemorrhage. Current biochemical and visual techniques lack the sensitivity and specificity to detect bilirubin at concentrations typically found in CSF following a bleed. In the direct spectral analysis of CSF, oxyhemoglobin greatly interferes with the absorption peak of bilirubin. This study attempts to use linear algebra to calculate bilirubin concentration in the presence of abnormal oxyhemoglobin and protein concentrations. The multiwavelength method was validated using a cohort of confirmed subarachnoid hemorrhage patients, as well a group of controls. A sensitive High Performance Liquid Chromatography method was developed for bilirubin as a gold standard reference method. Regression analysis showed that the multivariate method correlated well to the HPLC method. Receiver Operator Characteristic Curve analysis showed high specificity and sensitivity, suggesting future use of this assay as a tool in clinical diagnosis of subarachnoid hemorrhage.