New method for the determination of the net bilirubin absorbance in cerebrospinal fluid that minimizes the interference of oxyhaemoglobin and biliverdin.

The presence of oxyhaemoglobin and biliverdin interferes with the method recommended by the UK NEQAS Specialist Advisory group for EQA of CSF Proteins and Biochemistry for estimating of the net bilirubin absorbance in CSF. This is easily demonstrated by using solutions with different concentrations of these three substances.The two secondary peaks of the oxyhaemoglobin spectrum at 540 nm and 577 nm are used as reference to minimize these interferences. Those peaks have the same absorbance as at 456 nm in the oxyhaemoglobin spectrum, independent of its concentration. This wavelength is very close to the maximum absorption of bilirubin and, therefore, is suitable for estimating the net bilirubin absorbance.A preliminary study with 48 spectrophotometric analyses of CSF from patients who were suspected of having subarachnoid haemorrhage were used to compare both net bilirubin absorbance estimation methods.The new method is practically free of oxyhaemoglobin and biliverdin interference. This allows for higher sensitivity and a more realistic estimation of the bilirubin concentration in a sample.A better estimation of the bilirubin concentration can have special relevance for diminishing the amount of equivocal or inconclusive cases and also to improve the prematurity of the diagnosis.

Novel Biochemical Insights in the Cerebrospinal Fluid of Patients with Neurosyphilis Based on a Metabonomics Study.

Neurosyphilis is a chronic central nervous system infectious disease caused by Treponema pallidum. Our aim was to study the metabolic profiling in the cerebrospinal fluid of neurosyphilis patients and identify specific potential biomarkers. Fifteen cerebrospinal fluid samples from neurosyphilis patients and 14 non-neurosyphilis samples were analyzed by liquid chromatography-mass spectrometer (LC-MS). The LC-MS data were preprocessed by supervised pattern recognition to obtain diagnostic models. Both orthogonal projections to a latent structures discriminant analysis (OPLS-DA) and a t test were used to obtain specific metabolites for neurosyphilis. LC-MS data showed that the metabolites in cerebrospinal fluid (CSF) from neurosyphilis are different from the non-neurosyphilis group. The OPLS-DA model parameters R2Y and Q2Y are both more than 0.7 and indicated a satisfactory diagnostic performance. Bilirubin, L-histidine, prostaglandin E2, alpha-kamlolenic acid, and butyryl-L-carnitine and palmitoyl-L-carnitine were identified as novel potential biomarkers for neurosyphilis. The metabolic study of CSF may provide a new way to explore the pathogenesis of neurosyphilis.

Analysis of blood degradation products and ferritin in the cerebrospinal fluid of dogs with acute thoracolumbar intervertebral disk extrusion, a prospective pilot study.

BACKGROUND: Hemorrhage in the spinal canal leads to further damage of the spinal cord influencing outcome in dogs with intervertebral disk (IVD) extrusion. The aim of the study was to evaluate blood degradation products and ferritin in the cerebrospinal fluid (CSF) of dogs with thoracolumbar IVD extrusion, and their association to clinical parameters and MRI findings. RESULTS: In the CSF of dogs with IVD extrusion, both net oxyhemoglobin absorption (NOA) and net bilirubin absorption (NBA) were significantly higher compared to the control groups of dogs with steroid responsive meningitis arteritis (SRMA) and idiopathic epilepsy (IE) (P < 0.001), but NOA compared to the idiopathic epilepsy group contaminated artificially with blood (IEc) was not (P = 0.890). Ferritin concentration was significantly higher in dogs with IVD extrusion compared to dogs with IE (P = 0.034), but not to dogs with SRMA (P = 0.526). There was no association between NOA, NBA or ferritin concentration and severity or duration of clinical signs. In dogs with a higher ferritin concentration the outcome was better (P = 0.018). In dogs with evidence of hemorrhage on MRI, NOA and NBA were significantly higher (P = 0.016, P = 0.009), but not ferritin (P = 0.0628). CONCLUSION AND CLINICAL IMPORTANCE: Quantification of blood degradation products and ferritin in the CSF of dogs to assess subarachnoidal hemorrhage is feasible; however, larger case numbers are needed to evaluate the relevance of NBA and ferritin as prognostic indicators.

Froin Syndrome After Spinal Cord Injury.

BACKGROUND: Froin syndrome is characterized by xanthochromia and hypercoagulability of the cerebrospinal fluid (CSF) due to elevated protein levels. This entity results from blockage of the spinal canal by a mass lesion leading to an isolated caudal CSF space. CASE DESCRIPTION: A 48-year-old male, who developed spasticity after a C6 spinal cord injury (SCI) 20 years earlier, presented with subobstruction of his intrathecal baclofen pump. A catheter access port aspiration revealed an extremely high protein concentration (38 g/L) with no signs of infection. Froin syndrome was confirmed when magnetic resonance imaging showed a complete obstruction of the spinal canal at the SCI level. CONCLUSIONS: We report the first case of Froin syndrome after SCI. Froin syndrome can impact intrathecal drug delivery and CSF diagnostics.

Propentdyopents as Heme Degradation Intermediates Constrict Mouse Cerebral Arterioles and Are Present in the Cerebrospinal Fluid of Patients With Subarachnoid Hemorrhage.

RATIONALE: Delayed ischemic neurological deficit is the most common cause of neurological impairment and unfavorable prognosis in patients with subarachnoid hemorrhage (SAH). Despite the existence of neuroimaging modalities that depict the onset of the accompanying cerebral vasospasm, preventive and therapeutic options are limited and fail to improve outcome owing to an insufficient pathomechanistic understanding of the delayed perfusion deficit. Previous studies have suggested that BOXes (bilirubin oxidation end products), originating from released heme surrounding ruptured blood vessels, are involved in arterial vasoconstriction. Recently, isolated intermediates of oxidative bilirubin degradation, known as PDPs (propentdyopents), have been considered as potential additional effectors in the development of arterial vasoconstriction. OBJECTIVE: To investigate whether PDPs and BOXes are present in hemorrhagic cerebrospinal fluid and involved in the vasoconstriction of cerebral arterioles. METHODS AND RESULTS: Via liquid chromatography/mass spectrometry, we measured increased PDP and BOX concentrations in cerebrospinal fluid of SAH patients compared with control subjects. Using differential interference contrast microscopy, we analyzed the vasoactivity of PDP isomers in vitro by monitoring the arteriolar diameter in mouse acute brain slices. We found an arteriolar constriction on application of PDPs in the concentration range that occurs in the cerebrospinal fluid of patients with SAH. By imaging arteriolar diameter changes using 2-photon microscopy in vivo, we demonstrated a short-onset vasoconstriction after intrathecal injection of either PDPs or BOXes. Using magnetic resonance imaging, we observed a long-term PDP-induced delay in cerebral perfusion. For all conditions, the arteriolar narrowing was dependent on functional big conductance potassium channels and was absent in big conductance potassium channels knockout mice. CONCLUSIONS: For the first time, we have quantified significantly higher concentrations of PDP and BOX isomers in the cerebrospinal fluid of patients with SAH compared to controls. The vasoconstrictive effect caused by PDPs in vitro and in vivo suggests a hitherto unrecognized pathway contributing to the pathogenesis of delayed ischemic deficit in patients with SAH.

The effect of intestinal flora on the neural development of severe hyperbilirubinemia neonates.

OBJECTIVE: To investigate the effect of intestinal flora on the neural development of severe hyperbilirubinemia neonates. PATIENTS AND METHODS: The clinical data of 108 severe hyperbilirubinemia neonates admitted to the Dezhou People's Hospital from January 2015 through January 2018 were analyzed, and all newborns had a serum total bilirubin level > 342 µmol/L. Based on whether they suffered from neural development abnormalities, the neonatal patients were divided into the neural abnormality group (n=52) and the non-neural abnormality group (n=56). The unconjugated bilirubin levels in serum and cerebrospinal fluid (CSF) and the composition of intestinal flora were compared. RESULTS: Among 108 neonates, there were 55 cases with developmental abnormalities, in which 52 (48.13%) cases had neural developmental abnormalities, mainly epileptic patients. The serum and CSF unconjugated bilirubin levels of the neonatal patients in the neural abnormality group were (466.25±97.64) µmol/L and (9.64±2.98) µmol/L, respectively, which were higher than those in neonatal patients of the non-neural abnormality group [(357.89±72.53) µmol/L and (6.73±3.11) µmol/L], with statistically significant differences (p<0.05). The abundance of intestinal flora genus in the neonates in the neural abnormality group was lower than that in the non-neural abnormality group, and the comparisons of Fusobacterium, Catabacter, Succinivibrio, Clostridium and Bacteroides between the two groups showed statistically significant differences (p<0.05). DISCUSSION: The intestinal micro-ecological environment of newborns was vulnerable and easily affected by many factors such as methods of delivery, feeding ways and eating habits of their mothers. This study investigated the effects of intestinal flora on the neural development of neonates with severe hyperbilirubinemia. The results showed that, due to decreased intestinal flora diversity, the serum and cerebrospinal fluid bilirubin levels were elevated, and the abnormal rate of neural development was increased. CONCLUSIONS: Severe hyperbilirubinemia neonates with neural abnormalities have decreased diversity of intestinal flora genus and relatively high serum and CSF bilirubin levels, probably because the decrease in the diversity of intestinal flora genus leads to the change of the blood-CSF barrier permeability, leading to raised levels of bilirubin in serum and CSF, thus affecting the neural development of neonatal patients.

Usefulness of liver function tests in postmortem samples.

Liver function tests have been investigated in the forensic setting in several biological fluids collected at autopsy. Nevertheless, the results of these investigations have provided diverging information on postmortem stability of liver function markers and postmortem reliability of liver function assessment. The first aim of this study was to determine gamma-glutamyltransferase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, total proteins, and albumin in antemortem and postmortem serum samples in a series of cases that underwent forensic investigations and had both samples available. The second aim was to measure total bilirubin and gamma-glutamyltransferase in cerebrospinal fluid. Preliminary results indicated that femoral (and cardiac) blood postmortem serum concentrations of bilirubin, total proteins, and albumin, as well as femoral blood postmortem serum concentrations of gamma-glutamyltransferase, can be considered to reliably reflect antemortem serum concentrations, thus suggesting that postmortem values could be used as surrogates for antemortem levels. Measurable bilirubin levels were demonstrable in cerebrospinal fluid samples in both preservative-free and sodium fluoride tubes, though with levels varying widely and unpredictably, irrespective of liver disease severity.

Effect of Visible Light on Vasospasticity of Post-Subarachnoid Hemorrhage Cerebrospinal Fluid.

Background and Objective Cerebral vasospasm (CV) is a serious complication of subarachnoid hemorrhage (SAH) with high morbidity and mortality rates. The mechanism of CV has not been determined. There are many theories related to this unsolved issue, one of which supports CV as a two-stage phenomenon from a pathophysiologic perspective. The first stage consists of inhibition of neuronal nitric oxide synthase by oxyhemoglobin, which results in a decrease of nitric oxide (NO) production. The second stage consists of an increase in the levels of asymmetric dimethylarginine through bilirubin oxidation products (BOXes), which are oxidized by-products of hemoglobin metabolism. These in turn inhibit endothelial nitric oxide synthase (eNOS), which results in the blockage of the second NO production mechanism. BOXes are sensitive to visible light, as is their precursor bilirubin. The hypothesis of CV prevention using the photosensitivity of BOXes was tested in this study. Material and Methods Cerebrospinal fluid (CSF) obtained from two patients with SAH was divided in half and either exposed to a standard dose of visible light or not exposed to any light. The CSF was spectrophotometrically investigated and the concentration of BOXes was measured. A comparison between CSF samples exposed to light and not exposed to light was made. Using two groups of 16 rats each, the vasospastic effect of the CSF exposed and not exposed to light on arteries of the cortical surface was measured. The cortex was exposed using the cranial window. Results Spectrophotometric analysis revealed that the concentration of BOXes in the CSF decreased significantly after being exposed to visible light (p < 0.001). There was a significant difference of the vasospastic effect of CSF on exposed cortical arteries (p < 0.001). Conclusion The concentration of BOXes and the vasospastic effect of CSF taken from patients with SAH were significantly reduced after being exposed to visible light if compared with CSF not exposed to light.

Enriched administrative data can be used to retrospectively identify all known cases of primary subarachnoid hemorrhage.

OBJECTIVE: We derived and validated a method to screen all hospital admissions for 1° subarachnoid hemorrhage (SAH) by retrospectively implementing recognized diagnostic criteria. STUDY DESIGN AND SETTING: A screen for 1° SAH was developed using two previously created registries. Screen-positive cases underwent diagnosis confirmation with primary record review. A review of all patient hospital encounters with the diagnostic code for 1° SAH, and cross-referencing with an existing SAH registry was undertaken to identify missed cases. RESULTS: Three subscreens were combined to form the 1° SAH screen (sensitivity: 98.4% [95% CI: 91.7-99.7%], specificity: 93.4% [95% CI: 90.4-95.4%], n = 455 patients in validation sample). From 1,699 screen-positive admissions between July 1, 2002 and June 30, 2011, we identified 831 true cases of SAH of which 632 patients had 1° SAH from ruptured aneurysm/arteriovenous malformation (sensitivity: 96.5% [95% CI: 94.8-97.8%], specificity: 40.3% [95% CI: 38.1-42.6%]). A review of all encounters with a diagnostic code for 1° SAH yielded additional 22 true cases. CONCLUSION: When positive, our 1° SAH screen significantly increases the probability of this diagnosis in a particular hospitalization. The addition of patient hospitalizations encoded with the diagnostic code for 1° SAH improved sensitivity. Together, these methods represent the best way to retrospectively identify all cases of 1° SAH within an extensive sampling frame.

Clinical and diagnostic findings in patients with elevated cerebrospinal bilirubin.

INTRODUCTION: Cerebrospinal fluid (CSF) spectroscopy can identify subarachnoid haemorrhage (SAH) when CT is negative in patients presenting with acute severe headache. The primary objective of this study was to evaluate the clinical use and usefulness of CSF spectrophotometry. Secondary objectives were to identify other causes of elevated CSF bilirubin, to analyse headache descriptions and to compare clinical features in patients with an elevated CSF bilirubin among those with and without an intracranial vascular cause of SAH (avSAH). METHODS: Consecutive patients admitted to two hospitals in Enniskillen and Londonderry between 1 January 2004 and 30 September 2014 with CSF spectroscopy bilirubin results were identified from a clinical chemistry laboratory dataset. Patients with elevated CSF bilirubin were studied. Clinical demographics, delays to investigation and final diagnoses were recorded. Patients with avSAH were compared with patients without avSAH. RESULTS: Among 1813 patients with CSF spectrophotometry results, requests increased more than threefold during the study (p<0.001). Fifty-six patients had elevated CSF bilirubin. Ten (17.9%) had avSAH, of which 8 (14.3%) had aneurysmal SAH. Non-vascular causes of elevated CSF bilirubin included meningitis, spontaneous intracranial hypotension and carcinomatous meningitis. Headache descriptions varied. Time from headache onset to admission, CT scan and lumbar puncture did not differ significantly for patients with avSAH and non-avSAH. CSF red cell counts were higher among patients with avSAH than patients with non-avSAH (p=0.005). CONCLUSIONS: CSF bilirubin measurement has an important role in identifying avSAH in CT-negative patients presenting with a thunderclap headache. Better clinical selection of patients is required as CSF spectrophotometry, although sensitive, is not specific for SAH.

Defending a traditional practice in the modern era: The use of lumbar puncture in the investigation of subarachnoid haemorrhage.

INTRODUCTION: Acute severe headache is a common medical presentation, and a common area of diagnostic uncertainty. Subarachnoid haemorrhage (SAH) is the cause in a minority of patients and has a high rate of morbidity and mortality. Therefore, its conclusive diagnosis with computed tomography (CT) or lumbar puncture (LP) is paramount. With advancement in imaging technology, emerging evidence now suggests that LP is no longer required for a subset of patients as CT has 100% sensitivity in detecting SAH, when performed under specific conditions. OBJECTIVES: To assess the proportion of patients with conclusive CSF xanthochromia results following a negative CT scan in suspected SAH to determine the diagnostic efficacy of LP. METHODS: CSF bilirubin and oxyhaemoglobin spectrophotometric absorbance data from all centres in a regional health board were identified for consecutive patients over a 6-month period. Results were stratified as conclusive (positive or negative), or inconclusive according to national guidelines. RESULTS: 239 of 255 (93.7%) results were conclusive: 89.0% were negative (227 of 255). 4.7% of results were positive (12 of 255), revealing 4 cerebral aneurysms requiring treatment. 16 out of 255 (6.3%) samples were inconclusive, yielding 1 aneurysm requiring treatment. In the same period, there were 27 CT-positive cases of SAH. CONCLUSIONS: LP has a high diagnostic yield, eliminating the need for neurosurgical opinion or investigation in almost 90% of cases. The test is both cost and time efficient and subjects only a small number of patients to the radiation and contrast risks of angiography.

Specificity of elevated cerebrospinal fluid bilirubin in the investigation of subarachnoid haemorrhage.

BACKGROUND: The spectrophotometric examination of cerebrospinal fluid for bilirubin is an established investigation in patients with suspected subarachnoid haemorrhage. This study assesses the diagnostic specificity of an elevated cerebrospinal fluid bilirubin and how this may be influenced by the presence of oxyhaemoglobin and the concentration of cerebrospinal fluid total protein. METHODS: One thousand cerebrospinal fluid spectroscopy reports were reviewed. Electronic patient records were examined to determine the clinical outcome in patients with an elevated cerebrospinal fluid bilirubin. RESULTS: Forty-four out of 1000 cerebrospinal fluid scans showed an increase in cerebrospinal fluid bilirubin unrelated to elevated serum bilirubin concentrations. This was associated with subarachnoid haemorrhage in 16 (36%) cases. Subarachnoid haemorrhage was confirmed in 5/17 (29%) patients positive for cerebrospinal fluid bilirubin alone and in 11/27 (41%) patients positive for both cerebrospinal fluid bilirubin and oxyhaemoglobin. At cerebrospinal fluid total protein concentrations <1 g/L, the specificity for subarachnoid haemorrhage improved: 4/9 (44%) vs. 1/8 (13%) with an increase in cerebrospinal fluid bilirubin alone; 6/10 (60%) vs. 5/17 (29%) in patients with increases in both bilirubin and oxyhaemoglobin. CONCLUSION: While an increase in cerebrospinal fluid bilirubin may be considered consistent with subarachnoid haemorrhage, the likelihood that a subarachnoid haemorrhage has occurred is influenced by the presence of oxyhaemoglobin and the concentration of total protein in the cerebrospinal fluid.

Prognostic value of intrathecal heme oxygenase-1 concentration in patients with Fisher Grade III aneurysmal subarachnoid hemorrhage.

OBJECT: Experimental studies have demonstrated the crucial role of posthemorrhagic erythrocyte catabolism in the pathogenesis of subarachnoid hemorrhage (SAH). The authors of this study aimed to investigate the prognostic value of a series of CSF biomarkers linked to heme metabolism in SAH patients. METHODS: Patients with Fisher Grade III aneurysmal SAH undergoing early aneurysm obliteration were enrolled. The levels of heme oxygenase-1 (HO-1), oxyhemoglobin, ferritin, and bilirubin in intrathecal CSF were measured on the 7th day posthemorrhage. The associations of functional outcome with clinical and CSF parameters were analyzed. RESULTS: The study included 41 patients (mean age 59 ± 14 years; 16 male, 25 female), 17 (41.5%) of whom had an unfavorable outcome (Glasgow Outcome Scale score ≤ 3) 3 months after SAH. In terms of the clinical data, age > 60 years, admission World Federation of Neurosurgical Societies Grade ≥ III, and the presence of acute hydrocephalus were independent factors associated with an unfavorable outcome. After adjusting for clinical parameters, a higher level of HO-1 appeared to be the most significant CSF parameter related to an unfavorable outcome among all tested CSF molecules (OR 0.934, 95% CI 0.883-0.989, p = 0.018). Further analysis using a generalized additive model identified a cutoff HO-1 value of 81.2 µM, with higher values predicting unfavorable outcome (82.4% accuracy). CONCLUSIONS: The authors propose that the level of intrathecal CSF HO-1 at Day 7 post-SAH can be an effective outcome indicator in patients with Fisher Grade III aneurysmal SAH.

Cerebrospinal fluid total protein cannot reliably distinguish true subarachnoid haemorrhage from other causes of raised cerebrospinal fluid net bilirubin and net oxyhaemoglobin absorbances.

BACKGROUND: In cerebrospinal fluid (CSF) spectrophotometry, if the net bilirubin absorbance (NBA) and net oxyhaemoglobin absorbance (NOA) are both raised with a visible oxyhaemoglobin peak, the revised national guidelines for analysis of CSF bilirubin advise interpreting the results as 'Consistent with subarachnoid haemorrhage (SAH)' regardless of the CSF total protein concentration of the specimen. We wanted to study the range of CSF total protein concentrations found in confirmed SAH to establish if the CSF total protein value can give further guidance on the likelihood of SAH. METHODS: Consecutive cases from five different hospital sites were included if the CSF NBA was greater than 0.007 AU and the NOA was greater than 0.02 AU with a visible oxyhaemoglobin peak. For the cases identified, the laboratory information management system and patient records were interrogated to identify the total protein concentration of the CSF specimen and whether SAH had ultimately been confirmed or excluded by other methods and supporting evidence. RESULTS: Results from 132 patients were included. The CSF total protein range in confirmed SAH was 0.23-3.08 g/L with a median concentration of 0.7 g/L (n = 51). In the SAH excluded group, the CSF total protein range was 0.43-29 g/L with a median concentration of 1.9 g/L (n = 81). CONCLUSIONS: Although confirmed SAH was not associated with the very highest concentrations of CSF total protein, a definite CSF protein cut-off concentration above which SAH could reliably be excluded cannot be recommended.

Intraobserver and interobserver agreement in visual inspection for xanthochromia: implications for subarachnoid hemorrhage diagnosis, computed tomography validation studies, and the Walton rule.

BACKGROUND: Visual inspection for xanthochromia is used to diagnose subarachnoid hemorrhage (SAH), to validate computed tomography subarachnoid hemorrhage diagnosis and was used to determine the Walton rule. No study has assessed the reliability of xanthochromia. OBJECTIVE: To determine intraobserver and interobserver xanthochromia agreement. METHODS: Mock cerebrospinal fluid samples contained increasing concentrations of human oxyhemoglobin, bilirubin, and albumin. Non-color-blind observers randomly assessed samples against a white background twice under significantly differing illumination. Specimens were recorded as red, orange, yellow, or clear. RESULTS: Results were obtained for 26 observers (11 male, 15 female observers). We found that 19.2% of samples were misclassified: red, 11.7%; orange, 28.5%; yellow, 29.6%; and clear, 22.1% (χ = 213.2; P < .001). Of the yellow misclassifications, 88% were misclassified as clear. Female observers correctly classified samples significantly more frequently than male observers (P = .03). Intraobserver agreement differed significantly from expected for both male (χ = 105.6; P < .001) and female (χ = 99.9; P < .001) observers regardless of illumination. Interobserver agreement was poor regardless of sex (χ for male observers = 176.96, P < .001; χ for female observers = 182.69, P < .001) or illumination (χ for bright = 125.64, P < .001; χ for dark = 148.48, P < .001). Overall, there was 75% agreement in 46% of the tests and 90% agreement in only 36% of the tests. CONCLUSION: This simple laboratory study would be expected to maximize agreement relative to clinical practice. Although non-color-blind female observers significantly outperformed non-color-blind male observers, both intraobserver agreement and interobserver agreement for xanthochromia were prohibitively poor regardless of sex or illumination. Yellow was most frequently misclassified, 88% as clear (ie, true positives were commuted to false negatives). Xanthochromia is therefore highly unreliable for subarachnoid hemorrhage diagnosis and computed tomography validation. The Walton rule requires urgent clinical revalidation.

Multi-wavelength spectrophotometric analysis for detection of xanthochromia in cerebrospinal fluid and accuracy for the diagnosis of subarachnoid hemorrhage.

BACKGROUND: Cerebrospinal fluid (CSF) was examined for bilirubin, an important indicator for diagnosis of subarachnoid hemorrhage (SAH). METHODS: A multi-wavelength (340, 415, and 460 nm) spectrophotometric assay was developed for the quantitative measurement of bilirubin in CSF, enabling the mathematical correction for absorbance of hemoglobin and proteins. Bilirubin and hemoglobin results were correlated to HPLC and a standard colorimetric assay, respectively. A subset of samples was sent for an absorbance reading at 450 nm following baseline correction. The multi-wavelength bilirubin assay was validated on 70 patients with confirmed SAH and 70 patients with neurologic symptoms who ruled out for SAH. RESULTS: The multi-wavelength spectrophometric assay demonstrated no interferences due to proteins (albumin) up to 30 g/l or oxyhemoglobin up to 260 mg/l. The assay limit of detection was 0.2 mg/l, linear to 20 mg/l, and CVs ranged from 1 to 6% at bilirubin concentrations of 0.84 and 2.1mg/l. The spectrophotometric assay correlated to HPLC and the colorimetric assay for bilirubin and hemoglobin, respectively. Results also correlated to the absorbance method (with removal of samples with high hemoglobin and proteins). The area under the ROC curve for diagnosis of SAH was 0.971 and 0.954 for the HPLC and spectrophotometric assay, respectively. At a cutoff of 0.2mg/l, the clinical specificity was 100% for both assays, and the clinical sensitivity was 94.3% and 88.6% for SAH for the HPLC and spectrophotometric asays, respectively. CONCLUSIONS: The multi-wavelength spectrophotometric assay is an objective alternative to visual inspection, HPLC, and absorbance for CSF bilirubin.

Role of bilirubin oxidation products in the pathophysiology of DIND following SAH.

Despite intensive research efforts, by our own team and many others, the molecules responsible for acute neurological damage following subarachnoid hemorrhage (SAH) and contributing to delayed ischemic neurological deficit (DIND) have not yet been elucidated. While there are a number of candidate mechanisms, including nitric oxide (NO) scavenging, endothelin-1, protein kinase C (PKC) activation, and rho kinase activation, to name but a few, that have been investigated using animal models and human trials, we are, it seems, no closer to discovering the true nature of this complex and enigmatic pathology. Efforts in our laboratory have focused on the chemical milieu present in hemorrhagic cerebrospinal fluid (CSF) following SAH and the interaction of the environment with the molecules generated by SAH and subsequent events, including NO scavenging, immune response, and clot breakdown. We have identified and characterized a group of molecules formed by the oxidative degradation of bilirubin (a clot breakdown product) and known as BOXes (bilirubin oxidation products). We present a synopsis of the characterization of BOXes as found in human SAH patients' CSF and the multiple signaling pathways by which BOXes act. In summary, BOXes are likely to play an essential role in the etiology of acute brain injury following SAH, as well as DIND.