Transcript CSF Bilirubin Measurements on the Roche Modular

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 autoanalyser 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
Haemoglobin
Biliubin
570 600
415
450
540
Wavelength (nm)
570
S-test
5000
S-test (AU)
4000
3000
2000
1000
0
0
20
40
60
80
100
Haem index (mg/dL)
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)
Bilirubin - CSF assay (umol/L)
QC protocol
1.8
1.6
L2 - 1/5
1.4
L1 - neat
1.2
1
0.8
L2 - 1/10
0.6
0.4
L1 - 1/5
L1 - 1/10
0.2
0
0
2
4
6
8
10
12
14
16
18
Bilirubin - standard assay (umol/L)
20
0.06
30%
0.05
25%
0.04
20%
0.03
15%
0.02
10%
0.01
5%
0.00
0%
0
0.2
0.4
0.6
0.8
1
1.2
1.4
CV
SD
Precision Profile
1.6
Bilirubin (umol/L
• 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
Stdev
CV
Effect of haemolysis - Scanning
0.016
0.014
0.012
0.01
0.008
0.006
0.004
0.002
0
0.3
0.25
0.2
0.15
0.1
0.05
0
0
2
4
Haemoglobin (mg/dL)
6
NHA
NBA
• CSF with added haemolysate
• NBA falls as Hb increases
NBA
NHA
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
0.4
120
0.3
100
80
0.2
60
0.1
40
20
0
0
0
2
4
Haemoglobin (mg/dL)
6
Haem (AU)
Bilirubin (umol/L
• CSF with added haemolysate
• Measured Bilirubin unaffected
• Can quantitate haemoglobin with S-test
Bilirubin
S-Test
Patient Sample Comparison
12
Bili (umol/L
10
8
6
4
2
0
0
0.1
0.2
0.3
0.4
NBA
• 81 samples referred for CSF analysis
• Measured bilirubin v NBA
Patient Sample Comparison
CSF Bili (umol/L)
1
False Positives
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
False Negatives
0
0.005 0.01 0.015 0.02 0.025 0.03
NBA (AU)
• NBA <0.03 AU
CSF Bili (umol/L)
Patient Sample Comparison
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0.005 0.01 0.015 0.02 0.025 0.03
NBA (AU)
• Red dots: Haemoglobin > 1.2 mg/dL
Effect of Light
Bilirubin (umol/L)
2.5
S1-dark
2
S2-dark
1.5
S1-Fluoro
S2-Fluoro
1
S1-window
0.5
0
0:00
S2-window
1:00
2:00
Time (hours)
3:00
4:00
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