Transcript No Slide Title

AACB CSF Working Party Investigation of CSF Total Protein
Result Variation.
G.I.
1
Raines ,
G.
2
Whittaker ,
3
Jenkins ,
4
Tran ,
4
Kellerman ,
M.
S.
G.
M.
6
7
8
S. Jovanovich , T. Andersen , D.A. Berry .
1Clinical
5
Clough ,
J.
6
Gill ,
2Biochemistry,
Biochemistry Unit, The Alfred, Melbourne,Vic.3004, Australia;
Royal Prince Alfred Hospital, Sydney, Australia;
3Biochemistry Department, Austin Pathology, Austin Health, Heidelberg VIC., Australia; 4 Clinical Chemistry, Hunter Area Pathology Service,
5
6
Newcastle, NSW, Australia; Clinical Chemistry, ICPMR, Westmead Hospital, Westmead, NSW, Australia; RCPA-AACB Quality Assurance
7
8
Programs Pty Limited, Bedford Park, SA, Australia; Australian Scientific Enterprise, Sydney, Australia; SEALS Sutherland Centre of
Immunology, Sutherland Hospital, Caringbah, NSW, Australia.
Introduction
Results
Method (continued)
Results (continued)
Patient CSF samples 3, 8 and 9 were haemolysed. The
mean of Lab A and Lab B results were used and a ratio
was determined between this mean and the results of the
other three labs. The ratio for the RCPA QAP for Lab C =
1.09; Lab D = 1.37; Lab E =1.19. The ratio for the patient
CSF samples for Lab C = 1.09; Lab D = 1.37; Lab E
=1.14.
Table 3 Summary of the Total Protein results in g/L
Analyser
Analytical Principal
Reagent
A
Modular PPE
Turbidometric
Benzathonium chloride.
35uL
B
Architect ci8200
Turbidometric
Benzethonium Chloride
67.6 uL
C
Beckman
Coulter DXC
800
Colourimetric
Pyrogallol RedMolybdate
5uL
Fusion 5.1
Vitros Prot (CSF)
micro slide
Copper azide
10uL
Colourimetric
Pyrogallol Red
D
E
Dimension RXL
MAX
12 uL
Aim
The aim of the CSF working party is to improve the
quality of the analytical performance of the CSF total
protein assay. CSF total protein results from the RCPA
QAP show distinct assay groups. The CSF Working Party
conducted a survey of patient CSF samples to determine
the cause of this variation in the assay results.
Method
Three laboratories distributed twelve unfrozen patient CSF
samples. The following analysers were used: Roche
Modular PPE- Lab A; Abbott Laboratories Architect ci8200Lab B; Beckman Coulter DXC 800- Lab C; Ortho-Clinical
Diagnostics Vitros Fusion 5.1- Lab D and Siemens
Dimension RXL Max- Lab E.
Table 1 lists the analysers, reagent and methods used in
the study. They form the distinct total protein groups
identified in the RCPA QAP program.
Lab A
0.17
0.24
0.28
0.29
0.42
0.45
0.48
0.5
0.56
0.64
0.81
0.83
Lab B
0.18
0.21
0.27
0.28
0.44
0.43
0.47
0.53
0.61
0.66
0.89
0.86
Lab C
0.17
0.21
0.54
0.29
0.45
0.4
0.51
0.57
0.68
0.69
0.87
0.77
Lab D
0.22
0.32
0.94
0.45
0.63
0.51
0.62
0.75
1.05
0.89
1.05
0.97
Lab E
0.2
no result
0.91
0.34
0.52
0.45
no result
no result
0.83
no result
0.92
0.85
Conclusion
The following graphs summarise the results of the
RCPA QAP and patient CSF samples Lab E was not
able to assay four patient samples (indicated by green
arrows in Fig 2).
Figure 1 Graph of RCPA QAP CSF Samples vs. CSF TP (g/l)
Table 2. Summary of Calibration Material used in CSF total protein
Analysis.
Modular PPE
TP CSF
Calibrator
International
Reference
Preparation
No of
Calibrators
used
Measuring
Range of
undiluted
sample
Urine/CSF Protein
Calibrator
Architect ci8200
Abbott Laboratories
UPRO Cal
Standardized against
primary standard
traceable to NIST
SRM 927c
NIST SRM 927c
One vial-diluted to six
stds values on-board.
Five
Beckman
Coulter DXC 800
Synchron
Systems
MicroProtein
Calibrator
NIST National
Institute of
Standards and
Technology
One
Fusion 5.1
Dimension RXL
MAX
Vitros Calibrator 5
Randox Protein
(UP) calibrator
NIST SRM 92.7c
Internal Master
Lot of Calibrator
RCPA QAP CSF Samples vs CSF TP (g/L)
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
Three
1
2
3
4
5
6
7
8
0.1, 0.2, 0.4, 0.8, 2 g/L
1.0 g/L
0.1 - 3 g/L
9
Lab A
RCPA QAP Samples
0.04 - 2.0 g/L
10
Lab B
11
Lab C
12
Lab D
Lab E
0.0 - 1.5 g/L
Figure 2 Graph of Patient CSF Samples vs. CSF TP (g/l)
Concentrations
of Calibrators
g/L
These are lot specific.
Current lot is zero
blank and 2.07g/L
Analyte in
Calibrator
Albumin - human
serum; α-1
microglobulin - human
urine; IgG - human
serum; Total Proteinhuman serum and
sheep serum in lot
specific conc's
Abbott Laboratories
Urine / CSF Protein
70% human albumin :
30% human globulin
Urine (CSF)
Urine or Serum
Calibrator
Human serum, sheep
serum, human urine
Human
Ref Interval
0.15-0.45 g/L
0.15-0.40 g/L
0.01-2.00 g/L
0 - 3 g/L
0.12, 1.12, 2.9 g/L
0, 0.5 , 1.0 g/L
References
Patient CSF Samples vs CSF TP (g/L)
1. Brown P.D. et al. Neuroscience. 129(4) 2004: 955-968.
1.1
1
2. Daroriche R.O. et al. N Engl J Med. 355(19) 2006:
2012-2020.
0.9
Albumin and
globulin
Urine Calibrator
Albumin
Urine
Albumin
Urine
0.8
0.7
0.6
3. Franciotta D. et al. Journal of Neurological Sciences.
237 2005: 5-11.
0.5
0.4
0.3
0.2
< 0.45 g/L
0.12 - 0.6 g/L
Patient CSF total protein results showed the same
pattern of variability as seen in the RCPA CSF QAP
samples. Since a ratio was used we conclude that there
is no matrix effect contributing to the variation seen in the
RCPA QAP reports. Thus the groups of results can be
contributed to calibration or method variation and more
standardisation of assays is required to improve
comparability between laboratories.
The procedures and methods for determination of CSF
total protein are incompletely standardised and this is
reflected in the lack of agreement between laboratories .
Results of routine CSF total protein analysis is not
disease specific. The interpretation of the results should
take into account the sensitivity and specificity limitation.
0
Three
CSF TP (g/L)
CSF total protein levels are often used to support a
diagnosis. Elevated levels of CSF total protein are
suggestive of bacterial infection2. Suspected meningitis /
meningoencephalitis requires urgent CSF examination. It
has been stated that CSF total protein determination
should only be used as a screening test for meningitis3.
Laboratory
CSF Volume Required for TP assay
(uL)
CSF TP (g/L )
The composition of plasma and Cerebrospinal fluid (CSF)
are similar with the only major difference between the two
being the greatly reduced concentration of total protein in
CSF1. The CSF is actively secreted by the four choroid
plexuses and the concentration of some ions e.g. K+, are
independent of the variations in the plasma
concentration.
Sample
1
2
3
4
5
6
7
8
9
10
11
12
Table 1. Summary of Analysers and Methods.
<0.4 g/L
0.1
0
1
- MISSING SAMPLES
2
3
4
5
6
7
Patient Samples
8
Lab A
9
10
Lab B
Lab C
11
Lab D
12
Lab E