CSF Bilirubin Measurements on the Roche Modular: Better than Spectrophotometry? G Jones, M Roser...
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Transcript of CSF Bilirubin Measurements on the Roche Modular: Better than Spectrophotometry? G Jones, M Roser...
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