CSF Bilirubin Measurements on the Roche Modular: Better than Spectrophotometry? G Jones, M Roser Department of Chemical Pathology St Vincent’s Hospital, Sydney

Background CSF Bilirubin is an important test for excluding subarachnoid haemorrhage. The “Gold Standard” test is scanning spectrophotometry. Methods for measurements of bilirubin in CSF have been presented for Dade1 and Abbott2 analysers. We present a method for use on the Roche Modular system and illustrate benefits compared to spectrophotometry 1. Balsz et al; 2, Ungerer et al

CSF Bilirubin measurement Potential benefits of measurement on an auto-analyser compared to spectrophotometry: Does not require specialised equipment Does not require specialised training Requires a smaller sample volume Can be performed at all hours

But... Low bilirubin concentrations (<0.5 umol/L) Measurement of precision is difficult due to poor QC stability Haemoglobin interferes with many bilirubin assays Information from the presence of haemoglobin is not available for interpretation

Aims Establish and evaluate method for CSF bilirubin on the Roche Modular Precision Limit of quantitation Interference from haemoglobin Develop “Index” for haemoglobin in CSF samples for the Modular. Compare the effects of haemolysis on bilirubin estimation by spectrophotometry and Modular analyses.

Methods: Roche Bilirubin assay Roche Modular <P> analyser Roche TBILI Method (Cat 11822713) “New” Diazonium ion Sample volume 3 uL Limit of detection 1.7 umol/L Haemolysis – no interference up to 1000 mg/dL (no interference: greater of +/- 6.8 umol/L or 10%) Marked reduction in haemolysis interference compared to previous assay

Methods: CSF modifications Set up as user-defined chemistry with CSF application linked to routine serum application Used serum calibration protocols Sample volume increased 35u (11.6 fold increase) All samples for CSF protein get CSF bilirubin data available on fresh samples if Xanthchromia added later accumulation of data

Routine Measurement of Haem S-Test Set up on Serum Indices channel Primary wavelength 415 nm, secondary 480 nm Volume 8 uL; R1 200uL Reported in arbitrary units (absorption units) Measurement of Haem at 415 peak more sensitive than Roche H index and detects methaem as well as oxyhaem Called S-test as it detects Soret peak of haem

S-Test 415 480 570 600 Wavelength (nm) Haemoglobin Biliubin 415 450 570 600 415 450 570 540 Wavelength (nm)

S-test Good linearity with Haem index up to 80 mg/dL Reference interval < 20 AU (<0.8 mg/dL)

Precision Measurement Bilirubin measurements established using Modular “supernatant” and “urine” descriptors. Sample Vol: 3 uL, pre-diluted 1/10 (“supernatant”) and 1/5 (“urine”) Serum QC run on these channels as patients QC performed with Biorad serum QC BioRad L1 (15 umol/L) Biorad L2 (80 umol/L)

QC protocol Bilirubin - CSF assay (umol/L) Bilirubin - standard assay (umol/L)

Precision Profile SD (Pink squares) approx 0.3 – 0.4 umol/L CV (Blue diamonds) 20% at 0.25 umol/L Limit of detection (zero + 3 SD) 0.12 umol/L

Effect of haemolysis - Scanning CSF with added haemolysate NBA falls as Hb increases

Scanning Spectrophotometry: Example from Guidelines - normal Hb peak Oxy Hb peaks Bili peak Chalmers, Au, Clin Chem 2001;47:147-8 (South Australia)

Effect of haemolysis - Modular CSF with added haemolysate Measured Bilirubin unaffected Can quantitate haemoglobin with S-test

Patient Sample Comparison 81 samples referred for CSF analysis Measured bilirubin v NBA

Patient Sample Comparison False Positives False Negatives NBA <0.03 AU

Patient Sample Comparison Red dots: Haemoglobin > 1.2 mg/dL

Effect of Light

Conclusion The Modular CSF bilirubin assay is simple and robust. The S-test allow simultaneous measurement of CSF haem. The bilirubin assay is resistant to haemolysis improving clinical sensitivity compared to spectrophotometry. Clinical correlation remains to be confirmed but given the clinical requirements for sensitivity the use of this assay, we use this assay for routine rule-out purposes