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

2. 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

3. 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

4. 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

7. 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.

8. Methods: Roche Bilirubin assay
Roche Modular <P> analyser
Roche TBILI Method (Cat )
“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

9. 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

10. 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

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

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

13. 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)

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

15. 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

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

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

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

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

20. Patient Sample Comparison
False Positives
False Negatives
NBA <0.03 AU

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

22. Effect of Light

23. 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