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 subarachnoid haemorrhage from a traumatic lumbar puncture (LP) resulting in blood contamination of the CSF. While both in vivo haemorrhage and traumatic LP result in haemoglobin in the supernatant fluid, only in vivo haemorrhage results in bilirubin formation.
Following a true haemorrhage, red blood cells lyse and release oxyhaemoglobin, which is metabolised to bilirubin. The CSF supernatant after centrifugation is visibly pink or pink-orange due to oxyhaemoglobin, yellow due to bilirubin, and an intermediate colour if both are present together. The yellow colour due to bilirubin is the entity strictly defined as visible xanthochromia, as the term implies.
Chalmers and Kiley  published an improved method to determine xanthochromia in CSF which takes into account and compensates for the interference of haemoglobin and bilirubin absorption bands.
Figure 1: Molar Extinction Coefficient of Haemoglobin (left) and Bilirubin (right) [cm-1/M vs nm]
The method consists in scanning the region 350-550nm and printing the resulting spectrum. The analyst draws a tangent line from approx. 360nm to 530nm, which represents a corrected background of the spectrum. The height of haemoglobin and bilirubin bands is then measured by drawing two vertical lines between the corrected baseline and the apex of the corresponding peaks at 4 15nm and 440nm. Finally, the ratio of the two heights is calculated. For pure bilirubin, this ratio is 1, whereas for haemoglobin it is close to 8.
Although the Chalmers and Kiley method shows good correlation with angiography tests for the detection of subarachnoid haemorrhage, the process of drawing and measuring by hand the tangent baseline and peak heights introduces too large a subjective error.
Thanks to the advanced Band Area and Height Correction feature, Cecil Instruments spectrophotometers provide a fast, automated procedure to carry out this analysis with unparalleled ease, accuracy and reproducibility.
The method is demonstrated here using a holmium oxide filter as an example. In a real CSF test the same steps shall be followed, and only the wavelengths will be changed to conform to the ones specified above.
Press the SCAN key to access the Scan menu
Select b Store Baseline if a baseline has not been recorded, or a WL Scan Menu otherwise. The following prompts will be shown:
Note: Values in bold were entered by the operator during the example run. Values in brackets are adequate for real CSF analysis.
This produces the following holmium oxide spectrum:
Press the QUANT key to access the following menu:
Select a Quantitative Methods. The next menu will be displayed:
Finally select f Band Area Reprocess. The instrument prompts for the following parameters:
Note: Values in bold were entered by the operator during the example run. Values in brackets are adequate for re al CSF analysis.
The following graph is automatically generated in our example, with the baseline defined by the specified limits and the corrected height of the selected peak (H) automatically calculated:
Press the RUN/PRINT key to print the graph, or the NEXT key to proceed.
You are prompted to enter the parameters to calculate the second corrected height. Press ENTER to accept all the values introduced for the first band, except in Peak WL [nm] where you will enter the wavelength of the bilirubin band.
In our holmium oxide example:
The instrument displays the graph, and tabulates the results, for the second band:
The final operation is to calculate the ratio H415nm/H440nm and evaluate the results.
In our worked example,
the absolute absorbance of the first band at 445.5nm is 1.800A;
the absolute absorbance of the second band at 453.0nm is 0.731A;
the ratio H445.5/H453.0 is 2.46.
Typical CSF scans:
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