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Articles |
1
Department of Clinical Chemistry, Leiden University Medical Centre, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
a author for correspondence: fax 31-71-5266753, e-mail
froelandse{at}lumc.nl
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Introduction |
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 Top Introduction References |
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A solution to this problem was found in the mathematical step-by-step (iterative) unraveling of the main absorbance scan into the scans of the individual blood pigments via the iterative process.
On arrival at the laboratory, the CSF samples were centrifuged for 10
min at 1800g and ambient temperature. The supernatants were
kept in the dark to avoid degradation of bilirubin or were stored at
-20 °C. Before analysis, the pH of each supernatant was adjusted to
pH 6.6 (11). Absorbance scans were then made from 350 to 500
nm on a Beckman DU 640 spectrophotometer. The absorbances at
360, 405, 414, and 455 nm were recorded separately (see Fig. 1, A and B
).
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Concentrations of the blood pigments were then calculated with a
mathematical approximation technique (iteration procedure). In these
calculations (see below), the absorbances at 405, 414, and 455 nm,
which are the 
max of methemoglobin,
oxyhemoglobin, and bilirubin, respectively, were each corrected for the
absorbance of the other components. For this we used the relative
absorbances of these components at the three wavelengths and the
turbidity at 360 nm. The relative absorbance of component x
is defined as the ratio of the absorbance of a blood pigment
x at wavelength y
(Ay,x) and the maximum absorbance at
wavelength max
(Amax,x). We determined the
relative absorbances at 360, 405, 414, and 455 nm by measuring
solutions of pure methemoglobin, oxyhemoglobin, and bilirubin,
prepared according to the method of Stroes and van Rijn
(11); we used Intralipid (Pharmacia & Upjohn) solutions for
turbidity measurements. The absorbance at 360 nm was used as a measure
of interference by turbidity. In addition, the measured absorbances
were also corrected for a blank CSF sample for which we used fixed
absorbance values: 0.011, 0.008, 0.007, and 0.005 absorbance units at
360, 405, 414, and 455 nm, respectively. Repeating the iterative
process 20 times appeared to be sufficient in all cases.
For the calculation of, e.g., methemoglobin (m) in the first iteration,
the corrected absorbance (Am) was calculated
using the formula:
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where A405 is the absorbance at
405 nm, n is the iteration number (1–20), and o405 is the
relative absorbance of oxyhemoglobin at 405 nm. The same line of
reasoning was used for the other pigments [bilirubin (b),
oxyhemoglobin (o), and turbidity (t)]. The recalculated absorbances
for the individual pigments were then transformed to concentrations
using calibration curves. An example of the original scan of a CSF
sample is provided in Fig. 1A
. The absorbances contributed by the
individual compounds, as calculated by the iterative process, are
presented in Fig. 1B
.
The linearity of the method was investigated by increasing the concentration of one of the blood pigments in a solution containing high concentrations (3 µmol/L) of the other two pigments. In each case, when the calculated concentration was plotted against the expected concentration, we found a straight line with a slope close to 1 and an intercept that was nearly zero. The lower detection limits calculated from these lines were <0.1 µmol/L for all three pigments.
The reliability of the iterative calculation method was tested with
several mixtures of the four components in blank CSF. The results of
the added and calculated concentrations in 15 mixtures are presented in
Fig. 1C
. A close correlation was observed. In addition, we
supplemented 27 blank CSF samples with one or more of the blood
pigments. Concentrations were calculated with the computer program, and
eight experienced technicians interpreted the scans. The results are
presented in Table 1
, with the presence or absence of an added component used as the
"gold standard" to calculate positive and negative
predictive values, sensitivities, and specificities. Again, the
program calculated the concentrations of the pigments correctly,
although two results near the cutoff point of 0.1 µmol/L were
discrepant. Nevertheless, the results show that the performance of the
computer program is substantially better than the visual interpretation
by technicians.
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In another approach, using this computer program as the gold standard, 39 absorbance spectra of pathological and nonpathological CSF samples were visually examined by eight trained technicians. The calculated sensitivities [mean (range)] for methemoglobin, oxyhemoglobin, and bilirubin were 0.66 (0.35–1.00), 0.84 (0.54–1.00), and 0.83 (0.67–0.97), respectively. The specificities [mean (range)] were 0.79 (0.37–0.95), 0.42 (0.31–0.54), and 0.88 (0.67–1.00), respectively. There was a large variation in the results between the technicians, which can be overcome by use of the iterative procedure.
In summary, in this study we developed a simple, objective, quantitative, and technician-independent method for the interpretation of CSF spectra. The results show that interpretation of the spectra by individual technicians is subject to large intra- and interindividual variation, which complicates a useful clinical interpretation. Our calculation method bypasses these difficulties. However, even if the method is reliable, the results of CSF spectrophotometry must be interpreted carefully by the clinician because blood pigments can appear in CSF without any relation to a hemorrhage (2)(5)(6)(7)(10).
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Acknowledgments |
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References |
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TopIntroductionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  J. P. J. Ungerer, S. J. Southby, C. M. Florkowski, and P. M. George Automated Measurement of Cerebrospinal Fluid Bilirubin in Suspected Subarachnoid Hemorrhage Clin. Chem., October 1, 2004; 50(10): 1854 - 1856. [Full Text] [PDF]
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J. Egberts, B. van den Heuvel, H. J. Duiser, W. van Dam, E. G.W.M. Lentjes, and H. H.H. Kanhai Iterative, Spectrophotometric Method for Determination of Amniotic Fluid Bilirubin Concentrations: Comparison with the Liley Method Clin. Chem., November 1, 2002; 48(11): 2045 - 2047. [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), oxyhemoglobin (
), and bilirubin (
) in 15
mixtures of various compositions. Samples 1–10 did not contain
Intralipid (
). The final Intralipid concentrations in samples 11–15
were 27, 27, 7, 20, and 5 mg/L, respectively. The concentrations of the
three blood pigments are indicated on the left
y-axis and that of Intralipid on the
right y-axis.