Background - Visual assessment of cerebrospinal fluid (CSF) for xanthochromia ( yellow color) is practiced by the majority of laboratories worldwide as a means of diagnosing intracranical bleeds.Methods - Colorimetric and spectrophotometric analysis of CSF samples for recognizing the presence of bilirubin either in low concentrations or in the presence of hemolysed blood.Results - The experiments provide the physiological and colorimetric basis for abandoning visual assessment of CSF for xanthochromia.Conclusion - We strongly recommend relying on spectrophotometry as the analytical method of choice

Keir, G

Petzold, A

Sharpe, TL

English

UCL Discovery

Page - 1 -Author Information PageFull title: Why human color vision cannot reliably detect CSF xanthochromiaAuthors: Dr Axel Petzold MD PhD --- The Tavistock Intensive Care Unit, TheNational Hospital for Neurology and Neurosurgery & Department ofNeuroimmunology, Institute of Neurology, Queen Square, London WC1N 3BG,United Kingdom.Dr Geoffrey Keir PhD MSc FRCPath FIBMS --- The Department ofNeuroimmunology, Institute of Neurology, Queen Square, London WC1N 3BG,United Kingdom.Prof. Ted L Sharpe PhD MA --- The Colour and Vision Research Laboratories(http://cvrl.ioo.ucl.ac.uk/index.htm), Institute of Ophthalmology, 11-43 Bath Street,London EC1V 9EL, United Kingdom.Correspondence to: Dr Axel Petzold. The Department of Neuroimmunology, Instituteof Neurology, Queen Square, London WC1N 3BG, United Kingdom. Email:a.petzold@ion.ucl.ac.uk, Tel: +442078373611 extension 4204, Fax:+442078378553.Page - 2 -Acknowledgments and Funding PageWe thank Mr Robert Ludlow for excellent photographic support, Mr Miles Chapmanfor discussion and advice on the practical experiments and Prof EJ Thompson, DrGT Plant and Dr M Smith for comments on the manuscript.Conflict of interest statementNone declared.Financial interest statementNone declared.FundingNone.Page - 3 -Full title: Why human color vision cannot reliably detect CSF xanthochromiaCover title: XanthochromiaTables and Figures:1. Number of Tables: 12. Number of Figures: 1Key words: colour vision, spectralphotometry, xanthochromia, cerebrospinal fluidPage - 4 -AbstractBackground: Visual assessment of cerebrospinal fluid (CSF) forxanthochromia (yellow color) is practiced by the majority of laboratories worldwide as a means of diagnosing intracranical bleeds.Methods: Colorimetric and spectralphotometric analysis of CSF samples forrecognising the presence of bilirubin either in low concentrations or in the presenceof haemolysed blood.Results: The experiments provide the physiological and colorimetric basis forabandoning visual assessment of CSF for xanthochromia.Conclusion: We strongly recommend relying on spectrophotometry as theanalytical method of choice.Page - 5 -ArticleSubarachnoid haemorrhage (SAH) is one of the most striking conditions inmedicine with potentially fatal outcome. The analysis cerebrospinal fluid(CSF) is a crucial diagnostic tool. The sensitivity detecting a bleed by CT fallsfrom 95% on day one to <10% three weeks after the event [1], whilst thesensitivity of CSF analysis remains close to 100% [2].The presence of pigments in CSF alters its visual appearance.Oxyhaemoglobin makes it appear red or orange, whilst bilirubin gives theyellow coloration of true xanthochromia. Oxyhaemoglobin arises both from atraumatic tap and a true SAH. Importantly the conversion of oxyhaemoglobinto bilirubin can only happen in in vivo, allowing distinction between a trueintracranial bleed and one due to a traumatic tap. Here we providephysiological evidence that the commonly practised [3,4] visual assessment ofCSF should be abandoned and replaced by spectrophotometry.MethodsTo simulate the conditions in which bilirubin may be observed, a set ofexperiments were designed. Firstly, to reproduce contamination byoxyhaemoglobin as it may occur with a traumatic tap, a series of tubescontaining doubling dilutions of haemolysed blood (series 'A' inset in Fig 1A)into CSF containing the same amount of bilirubin was prepared. Secondly, todetermine the lowest concentration of bilirubin that could be confidentlydetected, we prepared a series of tubes containing doubling dilutions ofbilirubin alone (series 'B' inset in Fig 1A).All tubes were examined visually for xanthochromia, in normal daylight or coolwhite fluorescent light, the typical viewing conditions, by 11 analysts,comprising clinical scientists, biomedical scientists or clinical neurology staffwithin the Department of Neuroimmunology at National Hospital for Neurologyand Neurosurgery. The tubes were presented in a random order and thePage - 6 -analysts were naive to their actual concentrations.Once the visual assessments were complete, all samples were scannedbetween 350 and 740 nm using an Ultrospec 4300 pro (AmershamBiosciences). The same analysts were then asked to indicate whetherbilirubin was present or absent in the scan. The proportions of subjects findinga positive result by visual or spectroscopic assessment were compared usinga 2 test.Finally, the x-y chromaticity coordinates of the spectrophotometric scans werecalculated, according to the standard procedures of specification for visualassessment established by the Commission Internationale de l’Eclairage(CIE, the International Commission on Illumination). This involves multiplyingthe spectral transmittances of the samples, converted from their opticaldensities, by the spectral concentration of the radiant power of the sourceilluminating them and then multiplying the product by each of the three colormatching functions, which define the CIE standard colorimetric observer [5].The resulting x-y chromaticity coordinates of the samples can then be plottedin the CIE 1931 chromaticity diagram for the standard 2° field of view (Fig 1 A& B), and their dominant wavelengths, which correspond to hue, andexcitation purities, which correspond to saturation, can be geometricallycalculated (see explanations in the Table and Figure legends and values inTable 1). The calculations were made for two CIE illumination or lightingstandards: “D65,” which equates to average daylight; and “A”, which is fortungsten light.ResultsSamples in series ‘A’ , when calculated for viewing by standard illuminantD65, varied in their dominant wavelength from 572 nm (which falls near thehue category ‘pure yellow’ [6]) to 615 nm (red). They also varied in theirexcitation purity from 97.9% (highly saturated) to 34.1% (moderatelysaturated). In contrast, the samples in series ‘B’ all had the same dominantwavelength, 572 nm (yellow) but differed in their excitation purity from 0.62Page - 7 -(very desaturated) to 36.6% (Table 1). The optical density for bilirubin (450-460 nm) ranged from 3.5 to 0.36 for samples A1 to A14 and from 3.2 to 0.002for samples B1 to B8.A significantly higher proportion of the analysts detected traces of bilirubinspectrophotometrically than visually, both when the xanthochromic CSFsamples were contaminated by the presence of haemolysed blood (Series ‘A’)and when they were desaturated (Series ‘B’). In Series ‘A’, visual detectionfailed for CSF samples with dominant wavelengths greater than 574 nm(samples A1 – A7), most of which fall considerably outside the color category‘pure yellow’. In Series ‘B’, bilirubin could not be reliably detected in CSFspecimens with excitation purity levels below 2.4% (samples B5 – B8). Incontrast, in both Series ‘A’ and ‘B’, bilirubin could be reliably detected in all thesamples by examining the spectrophotometric scans.This study confirms that spectrophotometry is superior to color visionfor analysing CSF samples for the presence of bilirubin [7]. Most critical CSFsamples are either contaminated by oxyhaemoglobin or have only low levelsof bilirubin. Under such conditions, detection of xanthochromia becomesunreliable, especially when viewed under an incandescent lighting or atungsten desk lamp (corresponding to CIE standard illuminant A). Indeed, alower proportion of the assessors were able to detect xanthochromia forsamples 'B4' (7/11, 2=4.88, p<0.05), 'B5' (3/11, 2=9.21, p<0.01) and 'B6'(0/11, 2=6.47, p=0.01, see insets in Fig. 1B) under tungsten light than underdaylight conditions. Colorimetric analysis revealed that all of the samples nowfell completely outside the ‘pure yellow’ category. This condition represents a'worst-case scenario', such as may be encountered during a night on-call.DiscussionThe implications of these findings can be judged from our previous analysis ofspectrophotometric scans of CSF samples, which did not appear yellow inalmost 80% of the cases routinely encountered at the National Hospital forPage - 8 -Neurology and Neurosurgery [8].About 80% of all CSF samples with significant amounts of bilirubinappear rather 'reddish' than 'yellow' [8] but that 99.7% of over 3,500laboratories participating in two recent American surveys [3,4] still assesssamples by color vision. The observations presented here provide aphysiological basis for abandoning the visual assessment of CSF forxanthochromia.Page - 9 -References1. van Gijn J, van Dongen KJ. The time course of aneurysmal haemorrhage oncomputed tomograms.Neuroradiology. 1982;23:153-62. Vermeulen M, Hasan D, et al. The time course of aneurysmal haemorrhageon computed tomograms. J Neurol Neurosurg Psych 1989;52:826-83. Edlow JA, Bruner KS, Horowitz GL. Xanthochromia. Arch Pathol Lab Med.2002;126:413-54. Judge B. Laboratory analysis of xanthochromia in patients with suspectedsubarachnoidal hemorrhage: a national survey. In: Scientific Assembly, AmericanCollege of Emergency Physicians 2000. Philadelphia, PA5. Wyszecki G, Stiles WS. 1982 Color science: concepts and methods, quantitiativedata and formulae (2nd edition). John Wiley & Sons, New York6. Petzold A, Sharpe LT. Hue memory and discrimination in young children. VisionRes 1998;38:3759-727. Marden NA, Thomas PH, Stansbie D. Is the naked eye as sensitive as thespectrophotometer for detecting xanthochromia in cerebrovascular disease? In:Martin SM, ed. Proceedings of the National Meeting, 2001 April 30-May 4, London.London: Association of Clinical Biochemists, 2001: 538. Petzold A, Keir G, Sharpe LT. Spectralphotometry for xanthochromia. New Eng JMed 2004;351:1695-69. UK NEQAS For Immunochemistry Working Group. National guidelines for analysisof cerebrospinal fluid for bilirubin in suspected subarachnoid haemorrhage. Ann ClinBiochem 2003;40:481-8Page - 10 -Table legendTable 1. Color specifications (CIE 1931 x-y chromaticity coordinates, dominantwavelengths and excitation purities), calculated with respect to CIE illuminant D65, of14 samples derived by double diluting haemolysed blood (Series ‘A’) and 8 samplesderived from a double dilution of xanthochromic CSF in H2O (Series ‘B'). Thedominant wavelengths [3] were geometrically derived by drawing lines from thewhite-point (D65) through the x-y chromaticity coordinates of the samples to thecorresponding spectral wavelengths indicated on the outer boundary (spectral locus)of the chromaticity diagram (Figure 1). The excitation purities [3] are the ratios ofdistances in the chromaticity diagram, indicating how far the chromaticity points ofthe samples are displaced from the white-point towards the spectral locus. Alsoshown are the observed frequencies of assessors who correctly identified thepresence of bilirubin in the vision and spectrophotometry experiments and the resultsof the ² analysis.Page - 11 -Figure legendFigure 1 (A). A section of the CIE 1931 chromaticity diagram (the full diagram isshown in the lower left-hand corner) displaying the x-y chromaticity coordinates of 14samples simulating a traumatic tap derived by double diluting haemolysed blood intoxanthochromic CSF sample B1 (Series ‘A’, circles) and 8 samples derived from adouble dilution of xanthochromic CSF in H2O (Series ‘B’, triangles). The coordinateshave been calculated with respect to CIE illuminant D65 (white cross). Forreference, the x-y chromaticity coordinates and wavelengths of the spectral colorsfrom 550 nm (‘yellowish green’) to 620 nm (‘red’) are also indicated. The dominantwavelengths (turquoise lines) of samples 'B1-B8' and sample 'A1' are shown. Theregion corresponding to the color category 'pure yellow', which ranges between 575and 580 nm, is indicated in pale grey (see summary table in [4]). The photographicinsets show the Series ‘A’ and ‘B’ samples illuminated by cool white fluorescent light.(B). The x-y chromaticity coordinates of the 22 samples from Series ‘A’ (circles) and'B' (triangles) calculated for CIE illuminant A (white cross). The dominantwavelengths (turquoise lines) of samples 'B1-B8' and sample 'A1' are shown. Thephotographic inset shows Series ‘A’ and 'B' illuminated by a tungsten halogensource.Page - 12 -Table 1Sample Chromaticity-coordinates (x, y)Dominantwavelength (nm)Excitationpurity (%)Colour vision(observedfrequency)Spectrophotometry(observed frequency) ² p-valueA1 0.3824, 0.3990 615 97.9 0.00 0.72 12.57 <0.001A2 0.3828, 0.3993 607 91.2 0.00 0.72 12.57 <0.001A3 0.3836, 0.4002 596 77.3 0.00 0.72 12.57 <0.001A4 0.3847, 0.4011 586 64.3 0.00 0.72 12.57 <0.001A5 0.3868, 0.4030 580 55.4 0.09 0.72 9.21 <0.01A6 0.3903, 0.4058 576 48.0 0.18 0.90 11.73 <0.001A7 0.3961, 0.4097 574 42.8 0.63 1.00 4.88 <0.05A8 0.4052, 0.4138 573 39.5 1.00 1.00 -- N.S.A9 0.4209, 0.4166 572 36.7 1.00 1.00 -- N.S.A10 0.4478, 0.4142 572 36.0 1.00 1.00 -- N.S.A11 0.4922, 0.4000 572 34.8 1.00 1.00 -- N.S.A12 0.5619, 0.3709 572 34.6 1.00 1.00 -- N.S.A13 0.6354, 0.3383 572 34.3 1.00 1.00 -- N.S.A14 0.6769, 0.3180 572 34.1 1.00 1.00 -- N.S.B1 0.3883, 0.4052 572 36.6 1.00 1.00 -- N.S.B2 0.3615, 0.3702 572 18.2 1.00 1.00 -- N.S.B3 0.3474, 0.3518 572 9.3 1.00 1.00 -- N.S.B4 0.3403, 0.3425 572 4.8 0.90 1.00 -- N.S.B5 0.3366, 0.3378 572 2.4 0.45 1.00 8.25 <0.01B6 0.3348, 0.3354 572 1.66 0.00 1.00 22.0 <0.001B7 0.3338, 0.3341 572 1.04 0.00 1.00 22.0 <0.001B8 0.3334, 0.3336 572 0.62 0.00 1.00 22.0 <0.001Page - 13 -

Arch Pathol Lab Med.

Color science: concepts and methods, quantitiative data and formulae (2 nd edition).

For Immunochemistry Working Group. National guidelines for analysis of cerebrospinal fluid for bilirubin in suspected subarachnoid haemorrhage.

Hue memory and discrimination in young children.

Is the naked eye as sensitive as the spectrophotometer for detecting xanthochromia in cerebrovascular disease? In:

Laboratory analysis of xanthochromia in patients with suspected subarachnoidal hemorrhage: a national survey. In: Scientific Assembly, American College of Emergency Physicians

Spectralphotometry for xanthochromia.

The time course of aneurysmal haemorrhage on computed tomograms.

The time course of aneurysmal haemorrhage on computed tomograms.Neuroradiology.

Why human color vision cannot reliably detect cerebrospinal fluid xanthochromia

Why human color vision cannot reliably detect cerebrospinal fluid xanthochromia

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