Limitations of spectrophotometry in the analysis of cerebrospinal fluid bilirubin

BackgroundCerebrospinal fluid (CSF) spectrophotometry is well-established in the UK for measuring CSF bilirubin and oxyhaemoglobin to aid in the diagnosis of subarachnoid haemorrhage. Although it is known that some antibiotics and large amounts of oxyhaemoglobin interfere in the spectrophotometric analysis of CSF bilirubin, there is little published evidence about the impact of such interferences.MethodsExperimental samples were distributed to participants in the UK National External Quality Assessment Service (NEQAS) for Immunology, Immunochemistry and Allergy EQA programme for CSF haem pigments. Distribution 244 consisted of a pair of matched samples but with sample 244-2 containing an increased amount of oxyhaemoglobin compared to sample 244-1. Distribution 251 consisted of a pair of matched samples but with sample 251-2 containing additional doxycycline at a concentration of 0.5 µg/mL compared to sample 251-1. Participants analysed the samples spectrophotometrically and absorbance values were returned to UK NEQAS.ResultsThe net bilirubin absorbance (NBA) was significantly reduced in the presence of both interferants; adding 0.6% oxyhaemoglobin decreased the NBA by a mean of 55.9% and adding 0.5 µg/mL doxycycline decreased the NBA by a mean of 14.3%. Wilcoxon signed rank tests showed the NBA was significantly different for sample 2 compared to sample 1 for both distributions.ConclusionsIncreased amounts of oxyhaemoglobin and the presence of doxycycline can both negatively interfere with NBA. The interference is subtle and difficult to detect but has the potential to change a true positive result to a false negative, highlighting a significant limitation of the CSF spectrophotometry technique.