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.

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.

Cell-Free Oxyhemoglobin in Cerebrospinal Fluid After Aneurysmal Subarachnoid Hemorrhage: Biomarker and Potential Therapeutic Target.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (aSAH) is often complicated by the occurrence of delayed ischemic neurologic deficits (DIND), which impairs the clinical outcome of patients. The release of oxyhemoglobin (oxyHb) from lysing erythrocytes into cerebrospinal fluid (CSF) may critically contribute to the development of DIND. METHODS: Ventricular CSF of 18 high-grade (Fisher 3 and 4) aSAH patients was sampled daily from external ventricular drains between days 0 and 14 after bleeding. CSF was spectrophotometrically analyzed with precise quantification of cell-free oxyHb levels. RESULTS: OxyHb levels in CSF showed a delayed peak reaching the highest levels in the high-risk period for developing of DIND between days 3 and 14 after aneurysm rupture. Patients with DIND had a significantly higher cumulative oxyHb exposure within the first week after bleeding. CONCLUSIONS: OxyHb levels in CSF may be useful as a biomarker to predict DIND in aSAH patients. The contribution of oxyHb in CSF to the pathogenesis of DIND should be further investigated as a potential therapeutic target.

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.

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.

Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation.

BACKGROUND: Cerebral intraventricular hemorrhage (IVH) is a major cause of severe neurodevelopmental impairment in preterm infants. To date, no therapy is available that prevents infants from developing serious neurological disability following IVH. Thus, to develop treatment strategies for IVH, it is essential to characterize the initial sequence of molecular events that leads to brain damage. In this study, we investigated extracellular hemoglobin (Hb) as a causal initiator of inflammation in preterm IVH. METHODS: Using a preterm rabbit pup model, we investigated the molecular mechanisms and events following IVH. We also characterized the concentrations of cell-free Hb metabolites and pro-inflammatory mediators in the cerebrospinal fluid (CSF) of preterm human infants and rabbit pups. Finally, Hb metabolites were evaluated as causal initiators of inflammation in primary rabbit astrocyte cell cultures. RESULTS: Following IVH in preterm rabbit pups, the intraventricular CSF concentration of cell-free methemoglobin (metHb) increased from 24 to 72 hours and was strongly correlated with the concentration of TNFα at 72 hours (r2 = 0.896, P <0.001). Also, the mRNA expression of TNFα, IL-1ß, and Toll-like receptor-4 and TNFα protein levels were significantly increased in periventricular tissue at 72 hours, which was accompanied by extensive astrocyte activation (that is, glial fibrillary acidic protein (GFAP)staining). Furthermore, exposure of primary rabbit astrocyte cell cultures to metHb caused a dose-dependent increase in TNFα mRNA and protein levels, which was not observed following exposure to oxyhemoglobin (oxyHb) or hemin. Finally, a positive correlation (r2 = 0.237, P <0.03) between metHb and TNFα concentrations was observed in the CSF of preterm human infants following IVH. CONCLUSIONS: Following preterm IVH, increased metHb formation in the intraventricular space induces expression of pro-inflammatory cytokines. Thus, the formation of metHb might be a crucial initial event in the development of brain damage following preterm IVH. Accordingly, removal, scavenging, or neutralization of Hb could present a therapeutic opportunity and plausible approach to decreasing the damage in the immature brain following preterm IVH.

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.

An improved laboratory protocol to assess subarachnoid haemorrhage in patients with negative cranial CT scan.

BACKGROUND: The laboratory analysis of cerebrospinal fluid (CSF) plays a key role in considering subarachnoid haemorrhage (SAH) in patients with clinical suspicion, but negative CT scan. Although the determination of the CSF bilirubin concentration generally provides high sensitivity, it was recently shown that specificity and positive predictive value are unacceptably low, limiting its use as a diagnostic tool. METHODS: We intended to design and evaluate an improved laboratory protocol, which fulfills the requirement of better specificity without losing sensitivity. We present a procedure in which a "bili-excess" concentration is determined, which is the surplus CSF bilirubin measured after subtraction of an estimated upper limit for the individual patient. The latter is calculated from [bilirubin](serum), [albumin](serum) and [albumin](CSF), taking into account the propagation of analytical errors in the individual analyses. We investigated the applicability of direct absorption vs. derivative spectroscopy, thereby addressing the influence of various calibration methods. We evaluated our procedure in 92 CSF samples drawn from patients with (n=37) and without (n=55) clinical suspicion of SAH. RESULTS: In our study population, we show that specificity increases from 0.83 (95% CI, 0.74-0.91) to 1.00 (95% CI, 0.96-1.00) using the bili-excess concept, with an established upper limit for bili-excess of 0.11 micromol/L instead of the recommended use of an "uncorrected" CSF bilirubin upper limit of 0.20 micromol/L. Sensitivity in both cases is 1.00 (95% CI, 0.66-1.00). We demonstrate the merit of allowing for analytical imprecision in the bili-excess concept. CONCLUSIONS: We provide a quantitative procedure to explore the likelihood of (CT-negative) SAH independent of the absolute CSF bilirubin concentration by considering the "bili-excess" concentration per individual, using derivative spectroscopy to determine CSF bilirubin. This procedure led to an increase in specificity to 1.00 (95% CI, 0.96-1.00) in our study population.

Is cerebrospinal fluid spectrophotometry useful in CT scan-negative suspected subarachnoid haemorrage?

Missed cerebral aneurysms in CT-negative patients can have serious implications. We set out to determine the usefulness of cerebrospinal fluid (CSF) spectrophotometry and the individual significance of CSF oxyhaemoglobin, bilirubin and methaemoglobin in 463 CT scan-negative patients with suspected subarachnoid haemorrhage (SAH) and normal neurological examination. CSF spectrophotometry resulted in the diagnosis of an intracranial aneurysm in 2% (9/463) of patients with CT-negative suspected SAH. No aneurysms were found in patients in whom spectrophotometry was negative for haem pigments. Less than 1% of patients with oxyhaemoglobin alone had aneurysms diagnosed, whilst 21% of patients with bilirubin had an aneurysm. CSF spectrophotometry is an important investigation in patients with CT-negative suspected SAH, particularly where clinical suspicion is strong. Patients positive for bilirubin are likely to provide a high yield of aneurysmal bleed and should undergo angiography. Patients with oxyhaemoglobin alone in whom SAH is strongly suspected may benefit from angiography. Based on a small number of patients, we recommend that patients with methaemoglobin should also be investigated. Patients with negative spectrophotometry are unlikely to benefit from further investigation.

Suramin-induced reversal of chronic cerebral vasospasm in experimental subarachnoid hemorrhage.

OBJECT: The naphthylsulfonate derivative suramin is an inhibitor of growth factor receptors (receptor tyrosine kinases) and G protein-coupled P2Y receptors. Both types of these receptors are suspected of being involved in cerebral vasospasm after subarachnoid hemorrhage (SAH). In the current study, the authors examined the therapeutic effects of suramin and a selective P2X-receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), in the reversal of vasospasm in an established canine double-hemorrhage model. METHODS: Twenty-four dogs underwent double blood injection into the cisterna magna, with injections given on Days 0 and 2. The dogs were divided randomly into three groups (six animals in each group) to be treated from Days 2 through 6 with the vehicle dimethyl sulfoxide, suramin, or PPADS. An additional group of six dogs received double blood injection without any treatment and served as an SAH control group. The animals were killed on Day 7. Angiography was performed on Day 0 before blood injection and again on Day 7 before the animals were killed. After the death of the animals, the basilar arteries (BAs) were collected for morphological studies and determination of tyrosine kinase expression, and the bloody cerebrospinal fluid (CSF) produced by the hemorrhages was collected for measurement of oxyhemoglobin and adenosine triphosphate (ATP). In the SAH control group, the mean diameter of the BAs on Day 7 was 46.23 +/- 6.32% of the value on Day 0 (which served as a reference of 100%). In the DMSO-treated group, the mean residual diameter of the BA was 47.77 +/- 0.8% on Day 7 compared with the value on Day 0. Suramin, but not PPADS, increased the residual diameter to 74.02 +/- 4.24% on Day 7. On Day 7 the level of ATP in the CSF was decreased and the level of oxyhemoglobin was increased, compared with values measured on Day 0. Suramin, but not PPADS, reduced tyrosine phosphorylation in the spastic BAs. CONCLUSIONS: By reducing tyrosine kinase activity, suramin may be useful in the treatment of cerebral vasospasm.