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 570540450

Haemoglobin

Biliubin

Wavelength (nm)

480

570 600

415

S-test

0

1000

2000

3000

4000

5000

0 20 40 60 80 100

Haem index (mg/dL)

S-t

est

(AU

)

Good linearity with Haem index up to 80 mg/dLReference 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

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6 8 10 12 14 16 18 20

L1 - neat

L1 - 1/5L1 - 1/10

L2 - 1/5

L2 - 1/10

Bilirubin - standard assay (umol/L)

Bil

irub

in -

CS

F a

ssay

(um

ol/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

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Bilirubin (umol/L

SD

0%

5%

10%

15%

20%

25%

30%

CV Stdev

CV

Effect of haemolysis - Scanning

• CSF with added haemolysate• NBA falls as Hb increases

00.0020.0040.0060.008

0.010.0120.0140.016

0 2 4 6

Haemoglobin (mg/dL)

NB

A

0

0.05

0.1

0.15

0.2

0.25

0.3

NH

A NBA

NHA

Chalmers, Au, Clin Chem 2001;47:147-8 (South Australia)

Scanning Spectrophotometry: Example from Guidelines - normal

Hb peak

Bili peak

Oxy Hb peaks

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

0

0.1

0.2

0.3

0.4

0 2 4 6

Haemoglobin (mg/dL)

Bili

rubi

n (u

mol

/L

0

20

40

60

80

100

120

Hae

m (

AU

)

Bilirubin

S-Test

0

2

4

6

8

10

12

0 0.1 0.2 0.3 0.4

NBA

Bili

(u

mo

l/L

Patient Sample Comparison

• 81 samples referred for CSF analysis• Measured bilirubin v NBA

00.10.20.30.40.50.60.70.80.9

1

0 0.005 0.01 0.015 0.02 0.025 0.03NBA (AU)

CS

F B

ili (

um

ol/L

)

Patient Sample Comparison

• NBA <0.03 AU

False Negatives

False Positives

Patient Sample Comparison

• Red dots: Haemoglobin > 1.2 mg/dL

00.10.20.30.40.50.60.70.80.9

1

0 0.005 0.01 0.015 0.02 0.025 0.03NBA (AU)

CS

F B

ili (

um

ol/L

)

Effect of Light

0

0.5

1

1.5

2

2.5

0:00 1:00 2:00 3:00 4:00

Time (hours)

Bil

iru

bin

(u

mo

l/L

) S1-dark

S2-dark

S1-Fluoro

S2-Fluoro

S1-window

S2-window

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