Detection of Xanthochromia in Cerebrospinal Fluid,

Departments of Haematology and Neurology, Flinders University Medical Centre, Bedford Park, South Australia 5042, Australia
^a author for correspondence: Department of Haematology, Flinders University Medical Centre, Bedford Park, South Australia 5042, Australia e-mail Chalmers.AH{at}Flinders.edu.au

Lumbar puncture (LP) still has an important role to play in the diagnosis of subarachnoid hemorrhage. Although computed tomography (CT) scanning has replaced LP as the investigation of first choice, within 24 h of ictus 5% of cases will show no evidence of hemorrhage on CT scanning; this percentage is as high as 50% by 1 week, 30% after 2 weeks, and 0% after 3 weeks (1).

By definition, xanthochromia is the yellow discoloration indicating the presence of bilirubin in the cerebrospinal fluid (CSF) and is used by some to differentiate in vivo hemorrhage from a traumatic LP. In contrast to CT, CSF xanthochromia is present in all patients up to 2 weeks postictus and is still present in 70% of patients at 3 weeks (1)(2). A minimum period for CSF bilirubin detection is 12 h postictus (2). Thus, the detection of bilirubin in CSF appears to be the test of choice at late time points. Spectrophotometry of CSF in the visible region is, in general, considered more sensitive than visual examination, with peaks at 415 and ~440–460 nm indicating the presence of hemoglobin (Hb) and bilirubin, respectively (1)(2). The problem with this test is that it is not known which CSF bilirubin absorbance indicates a clinically significant bleed. We have developed a simple quantitative method that attempts to address this question.

CSF was collected aseptically by LP for routine biochemical and microbiological investigations. The supernatants from CSF specimens microcentrifuged at 13 000g for 1 min were scanned between 360 and 800 nm in a spectrophotometer (Model 7500, Beckman Instruments). In most CSF samples, volumes collected were <500 µL and were scanned in 100-µL microcuvettes with a 1-cm light path. Volumes 500 µL were scanned in 1.5-mL cuvettes. After a scan was completed, the spectrum was autoscaled, and tangents were drawn from ~530 to 360 nm (Fig. 1 ). Perpendiculars to this tangent were measured in mm at 415 nm for Hb (a) and 440 nm for bilirubin (b). For pure bilirubin, this ratio of a/b was 1, whereas for Hb it was 8. In our scans, full-scale absorbance was represented by an 82-mm scale; hence the absorbance of Hb was a/82 multiplied by the full-scale absorbance, and the absorbance of bilirubin was b/82 multiplied by the full-scale absorbance. Because Hb increases the absorbance of bilirubin at 440 nm, the following correction to bilirubin absorption was made:

View larger version (24K): [in this window] [in a new window]   Figure 1. Spectrophotometric scans on CSF specimens from six patients with suspected intracranial hemorrhages. Drawn tangents (vertical lines) are the absorbances at 415 and 440 nm.

where 7 is 8 (a/b ratio for pure Hb) minus 1 (a/b ratio for pure bilirubin). Thus (8 - a/b)/7 allows for the contribution of the Hb absorbance at 415 nm to the bilirubin absorbance at 440 nm. The net bilirubin absorbance was used to quantify the xanthochromia.

In this study we looked at three groups: (a) controls representing 16 patients who underwent elective CT myelography, (b) patients considered positive xanthochromia with net bilirubin absorbances >0.015, and (c) patients with equivocal values with net bilirubin absorbances from 0.005 to 0.014.

In our control samples, net bilirubin absorbances ranged from 0 to 0.007, with a mean of 0.0023. The five patients with absorbances >0.015 all had subarachnoid hemorrhage: four because of aneurysmal rupture (proven angiographically) and one because of severe head injury. In our experience, at least three of these patients had a negative result for xanthochromia by direct inspection. Hb absorbances of these specimens, with net bilirubin absorbances in parenthesis, were 0.022 (0.022), 0.047 (0.026), 1.27 (0.93), 0.22 (0.18), and 0.2 (0.048). Thus, the Hb absorbances were variable and in four of the five samples were no different to those found in control specimens (range, 0–0.35; mean, 0.077 ± 0.1 SD).

Seven patients had net bilirubin absorbances between 0.005 and 0.014. Of these, four showed no abnormalities present by CT head scans and cerebral angiography. The other three did not undergo angiography, but were considered by a consultant neurologist to have migraine. At this time, we report values between 0.01 and 0.015 as borderline positive and values >0.015 as positive.

The scans of six patients shown in Fig. 1 were typical of the scans obtained. In patients A and C, considerable Hb was present, but only patient C was reported and confirmed clinically as positive for xanthochromia. With patient B, the 415/440 ratio was close to unity, indicating almost pure bilirubin, but the absorbances were low enough to consider this patient's result as being negative. In patients D, E, and F, although the absorbances of Hb were similar (0.022), the net bilirubin absorbances and consequent clinical reports were different: 0.006 (negative), 0.003 (negative), and 0.022 (positive), respectively.

The report that one can use a CSF Hb absorbance at 415 nm of >0.023 as a possible index of intracranial hemorrhage (3) is not tenable. For example, in one of our positive samples, the absorbance at 415 nm was 0.022. In addition, the finding that CSF proteins >1 g/L can produce absorbances >0.023 at 415 nm would suggest that absorbance measurements solely at 415 nm would yield low sensitivity in relation to determining xanthochromia (4). It makes good sense, therefore, to do a full spectral scan because it is the spectral hump between 440 and 460 nm that is clinically significant for xanthochromia.

One CSF specimen kept at room temperature (23 °C) for 48 h gave absorbances for Hb of 0.03, 0.09, and 0.31 at 0, 24, and 48 h, respectively. The absorbance due to bilirubin was 0 over these 3 days, suggesting that CSF does not need to be analyzed immediately, because there was no detectable alteration of Hb to bilirubin within the CSF after collection. This is consistent with the finding that Hb requires the enzyme hemoglobin oxidase present in macrophages within the arachnoid and choroid plexus to be converted to bilirubin (5).

Addition of bilirubin (Sigma Chemical Co.) in increasing amounts to CSF (net bilirubin absorbances, 0.024–0.3) yielded a recovery of bilirubin calculated by this method of 98.2% (± 6.0%, n = 9). A quality-control sample was prepared by diluting serum 100-fold in water and adding a known amount of bilirubin. The interday CV of this quality-control sample analyzed by three separate analysts 13 times over a 1-month period was 6.0% (mean net bilirubin absorbance, 0.0346 ± 0.0021). The intraday CV was 3.3% on this sample (mean absorbance, 0.0377 ± 0.00123, n = 12).

The correlation between serum bilirubin and CSF bilirubin was -0.058 (n = 31), indicating that the CSF bilirubin absorbance was not because of diffusion of bilirubin into the CSF or a consistent contamination of the CSF with vascular blood during the LP. In all our positive results to date, the clinical follow up has confirmed positive xanthochromia.

In conclusion, this test is simple, inexpensive, can easily be interpreted, and thereby overcomes the confusion in diagnosing subarachnoid hemorrhage (6)(7)(8). We have used it for the last 20 months in over 50 patients, and it has proven clinically useful.

References

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