Geoff Keir

A specific and sensitive ELISA for measuring S-100b in cerebrospinal fluid.

A sensitive, simple and specific sandwich ELISA for S-100b is described. This method involves the binding of a monoclonal anti-S-100b antibody to the wall of a microtitre plate. This capture antibody is subsequently incubated with S-100b standard, control or patient sample in the form of cerebrospinal fluid (CSF). After incubation, the microtitre plate is washed and horseradish peroxidase-labelled polyclonal anti-S-100b is added (detector antibody). The amount of detector antibody bound to the microtitre plate is proportional to the amount of S-100b in the sample. The assay has a lower limit of detection of 0.04 ng/ml and shows < 0.006% reactivity with the closely related polypeptide S-100a. The assay has a mean within-batch precision of 9.3 and 5.6% at S-100b concentrations of 0.38 and 0.8 ng/ml, respectively. The between batch precision is 8.9 and 8.1% at S-100b concentrations of 0.12 and 0.34 ng/ml, respectively. The recovery of S-100b from CSF spiked with 0.5 ng/ml was 94% with a CV of 8.5%. The assay may be completed in less than 5 h using precoated microtitre plates, thus lending itself to routine use in clinical laboratories. Using this ELISA, 154 CSF samples were analysed and 19% of samples were found to have elevated levels. The highest levels were found in patients with cerebral haemorrhage or central nervous system malignancy. S-100b concentrations from individuals without evidence of neurological disease were found to be less than 0.4 ng/ml. Only 5% of patients with multiple sclerosis were found to have elevated CSF S-100b concentrations. Serial CSF samples taken from a patient with an infected in-dwelling shunt showed a dramatic decline, suggesting that S-100b is rapidly cleared.

Soluble E-selectin in multiple sclerosis: raised concentrations in patients with primary progressive disease.

OBJECTIVE--To determine whether concentrations of soluble E-selectin (sE-selectin), an immunological marker of endothelial activation, were correlated with gadolinium-DPTA enhancement on MRI in patients with multiple sclerosis. METHODS--Serial sE-selectin concentrations were measured in 28 patients with multiple sclerosis undergoing monthly gadolinium (Gd) enhanced MRI of the brain and spinal cord, and in 10 control subjects. C reactive protein (CRP), von Willebrand factor (vWF), and tumour necrosis factor-alpha (TNF alpha) were also determined. RESULTS--Primary progressive patients had significantly increased sE-selectin concentrations compared with the relapsing remitting and secondary progressive patients who had normal sE-selectin concentrations (22.2 (SD1 6.1) ng/ml v 9.8 (SD2.1) ng/ml and 7.7 (SD2.7) ng/ml, respectively, P = 0.03). This difference was attributable to five of the 10 primary progressive patients who had persistently raised sE-selectin concentrations, with relatively inactive MRI studies. No correlation could be found between sE-selectin concentrations and Gd enhancement on MRI, but a close correlation existed between mean concentrations of sE-selectin and TNF alpha (r = 0.71, P < 0.001). Despite raised sE-selectin and TNF alpha concentrations, primary progressive patients had normal CRP concentrations (1.03 (SD1.14) mg/l), which were significantly lower than the relapsing remitting (3.16 (SD2.54) mg/l) and secondary progressive patients (2.28 (SD2.1) mg/l, P = 0.03). Raised CRP concentrations did correlate with infectious episodes, clinical relapse, and Gd enhancement, and were significantly raised when no MRI activity was found. Concentrations of vWF were normal in all patient groups. CONCLUSIONS--The results further high-light the differences between patients with primary progressive and those with relapsing remitting/secondary progressive multiple sclerosis.

Alterations in Cerebrospinal Fluid Apolipoprotein E and Amyloid β-Protein after Traumatic Brain Injury

There is evidence that apolipoprotein E (apoE) and amyloid beta-protein (Abeta), which are implicated in the pathology of chronic neurodegenerative disorders, are involved in the response of the brain to acute injury; however, human in vivo evidence is sparse. We conducted a prospective observational study to determine the magnitude and time-course of alterations in cerebrospinal fluid (CSF) apoE and Abeta concentrations after traumatic brain injury (TBI), and the relationship of these changes to severity of injury and clinical outcome. Enzyme linked immunosorbant assay (ELISA) was used to assay apoE, Abeta(1-40) and Abeta(1-42) in serial CSF samples from 13 patients with TBI and 13 controls. CSF S100B and tau were assayed as surrogate markers of brain injury. There was a significant decrease in CSF apoE (p < 0.001) and Abeta (p< 0.001) after TBI contrasting the observed elevation in CSF S100B (p < 0.001) and tau (p < 0.001) concentration. There was significant correlation (r = 0.67, p = 0.01) between injury severity and the decrease in Abeta(1-40) concentration after TBI. In vivo, changes in apoE and Abeta concentration occur after TBI and may be important in the response of the human brain to injury.

Temporal Alterations in Cerebrospinal Fluid Amyloid β-Protein and Apolipoprotein E After Subarachnoid Hemorrhage

Background and Purpose— The mechanism underlying the association between possession of the APOE &egr;4 allele and less favorable outcome after subarachnoid hemorrhage (SAH) remains to be determined. After SAH the level of apolipoprotein E (apoE) in the cerebrospinal fluid (CSF) is decreased, and lower levels are associated with more severe injury and less favorable outcome. This study examined serial CSF samples to determine the time course for the decrease in CSF apoE and the relationship between CSF apoE and amyloid &bgr;-protein (A&bgr;), testing the hypothesis that apoE-A&bgr; interactions occur in vivo after SAH. Methods— Enzyme-linked immunosorbent assay was used to assay apoE, A&bgr;1–40, and A&bgr;1–42 in serial ventricular CSF samples from 19 patients with SAH and 13 controls. CSF S100B and &tgr; were assayed as surrogate markers of brain injury. Results— There was a sustained decrease in CSF apoE (P <0.001) and A&bgr; (P <0.001) after SAH in contrast to the observed elevation in CSF S100B (P <0.001) and &tgr; (P <0.001) concentration. There was significant correlation between CSF A&bgr; concentration and clinical outcome (r =0.65, P <0.01), and the decrease in CSF A&bgr; concentration correlated significantly with that of apoE (r =0.85, P <0.0001). Conclusions— After SAH both apoE and A&bgr; levels decrease in the CSF, supporting the concept that interactions between these proteins occur in vivo. The possibility that apoE and A&bgr; influence outcome after SAH warrants further investigation.

The clinical significance of an intrathecal monoclonal immunoglobulin band: a follow-up study.

To the Editor: With modern CSF analysis, oligoclonal bands can be demonstrated in the CSF of patients with clinically definite multiple sclerosis (MS) at a rate of 95%.1 This may be closer to 100% if the cut-off was lowered to one band but there may be an impact on specificity. Davies et al.2 have addressed this question, since the existing literature on the significance of a single band focuses on work conducted with agarose-electrophoresis, which is of low resolution and sensitivity. However, some issues need clarification. What was the overall frequency of a single band in the whole population? Restriction to the 31 patients who consented to repeat lumbar puncture might introduce a bias toward patients with demyelinating diseases. How many bands were present in the follow-up CSF and was the change to an oligoclonal pattern accompanied by a quantitative increase in intrathecal immunoglobulin G (IgG)? Considering the unavoidable variance (VK?) between different gels, it is advisable to rerun the original (frozen) specimens along with the follow-up samples. Furthermore, faint bands might escape detection when CSF and sera are not diluted to exactly the same uniform IgG concentration. It is important to note that detection of total IgG is less sensitive than, for example, affinity immunoblotting. Sindic et al.3 have shown that the oligoclonal IgG does not coincide with the oligoclonal staining for various microbial antigens and negative staining for IgG does not rule out oligoclonal reactions against such antigens. Thus, what might appear as a single IgG band may still be oligoclonal. The interchangeable use of monoclonal and single band is confusing. The term monoclonal bands (plural) is already reserved for the type 5 pattern (IgG paraprotein) as defined in the European consensus.1 Although a (truly) monoclonal IgG paraprotein will appear as a single band in agarose-electrophoresis, it is split up in 2–15 bands (own observations) in isoelectric focusing because of postsynthetic modifications. The number of bands alone does not allow differentiation between oligoclonal and monoclonal. Many patients with a single band and no obvious explanation for intrathecal IgG synthesis speak against a cut-off of one band. In order to retain high sensitivity and specificity, I suggest the use of two cut-off levels: two to three bands rated as borderline and four or more as definitely positive.4

Decreased Cerebrospinal Fluid Apolipoprotein E After Subarachnoid Hemorrhage. Correlation With Injury Severity and Clinical Outcome

Background and Purpose— The apolipoprotein E (APOE) &egr;4 allele has been associated with unfavorable outcome after subarachnoid hemorrhage (SAH), suggesting that apoE plays an important role in the response of the brain to SAH. We determined the concentration of apoE in the cerebrospinal fluid (CSF) of patients with SAH and a control group to test the hypothesis that alterations in CSF apoE reflect the response of the brain to SAH and are correlated with the severity of injury and outcome. Methods— ApoE and S100B (a marker of brain injury) were measured by ELISA in CSF from a non–brain-injured control group and patients with SAH. The severity of SAH was determined from the Glasgow Coma Scale, and the clinical outcome was determined from the Glasgow Outcome Scale. Results— In contrast to increased CSF concentration of S100B, CSF apoE concentration was significantly lower in patients after SAH than in control subjects (Mann-Whitney test, P <0.0001). SAH patients with more severe injury and less favorable outcome had lower CSF apoE concentration than did patients with milder injury and better clinical outcome (Fisher exact test, P =0.02). Conclusions— The concentration of apoE in the CSF decreases after SAH, despite the likely leakage of plasma apoE into the CSF. We speculate that apoE is retained within the parenchyma of the central nervous system in response to injury, where, in view of previous data, it may have a protective role.

Cerebrospinal fluid apolipoprotein E concentration decreases after traumatic brain injury

The APOE epsilon4 allele has been associated with unfavorable outcome after several types of acute brain injury, yet the biological mechanisms underlying this observation are poorly understood. Postmortem and experimental brain injury studies suggest the presence of increased amounts of apolipoprotein E (apoE) within the neuropil after acute brain injury. We assayed the concentration of apolipoprotein E in the cerebrospinal fluid (CSF) of non-injured controls and patients with traumatic brain injury (TBI) to determine whether differences exist, and if these differences correlate with injury severity and clinical outcome. CSF apoE and S100B, a marker of injury severity, were measured by enzyme linked immunosorbant assay. CSF was sampled from 27 traumatic brain injury patients (mean age 32, median 25, range 16-65 years) within 3 days of injury, and 28 controls (mean age 40, median 37, range 19-73 years). The TBI patients all had a Glasgow Coma Score (GCS) of less than eight (i.e., severe head injury). Clinical outcome was determined using the Glasgow Outcome Score (GOS). The average concentration of apoE in the CSF of controls was 12.4 mg/L (95% CI: 10.5-14.3 mg/L) and in TBI patients was 3.7 mg/L (95% CI: 2.1-4.1 mg/L; Mann-Whitney: p < 0.0001). In contrast, the concentration of S100B in the CSF of TBI patients was significantly higher than that of controls (Mann-Whitney: p < 0.0001). We speculate that apoE is retained within the parenchyma of the central nervous system in response to injury where in view of previous data, it may have a protective role.

Rostrocaudal Dynamics of CSF Biomarkers

The rostrocaudal gradient (RCG) of markers present in cerebrospinal fluid (CSF) has not been studied adequately due to lack of appropriate control populations and ethical restrictions. The aim of this study is to understand the rostrocaudal gradient of CSF biomarkers. We contacted a study comparing CSF levels of seven biomarkers from cisternal (rostral) and lumbar (caudal) CSF obtained from patients with trigeminal neuralgia and tension-type headache. The RCGs of CSF/serum albumin ratio, 8-isoprostane. GFAP, total tau and beta amyloid protein were higher than one. The RCGs of lactate, VEGF and the heavy chain of neurofilament protein were lower than one. The study provides new values for several commonly examined markers of cisternal CSF. Knowledge of the RCG gradient of different CSF markers is important in interpreting studies reporting ventricular CSF values.

Use of cerebrospinal fluid amyloid-β and total tau protein to predict favorable surgical outcomes in patients with idiopathic normal pressure hydrocephalus

OBJECT The prognostic value of CSF biomarkers in patients with idiopathic normal pressure hydrocephalus (iNPH) has not been adequately studied to date. The aim of this study was to identify CSF markers of favorable surgical outcome in patients with iNPH undergoing the insertion of a ventriculoperitoneal shunt. METHODS Ventricular CSF was collected intraoperatively from 22 patients with iNPH and enzyme-linked immunosorbent assay was used to analyze the levels of amyloid-β 1-42 (Aβ(1-42)) and total tau protein. The Black grading scale was used to assess outcomes at 6 months. Receiver operating characteristic (ROC) curves were obtained and discriminant function analysis was undertaken to provide sensitivity and specificity figures for each marker as well as their combination. RESULTS The mean age of the patients was 71.45 years (± 9.5 years [SD]). Follow-up was achieved in 21 patients. Seventeen patients had a favorable outcome and 4 patients had unfavorable outcome at 6 months. An Aβ(1-42) level of 180 pg/ml had a sensitivity of 35% and a specificity of 20% for predicting a favorable outcome at 6 months. A total tau level of 767 pg/ml will have a sensitivity of 17% and a specificity of 20% for predicting a favorable outcome at 6 months. A combination of Aβ(1-42) and total tau levels predicted favorable outcomes with a sensitivity of 80% and specificity of 82.4%. CONCLUSIONS In this pilot study a combination of Aβ(1-42) levels and total tau protein levels predicted favorable surgical outcomes at 6 months with adequate accuracy to be of clinical use. Further study in a larger group with longer follow-up is warranted.

Protein adsorption to hydrocephalus shunt catheters: CSF protein adsorption

OBJECTIVE To assess the quantity and nature of the proteins that adsorb to hydrocephalus shunt catheters after implantation, and to determine whether sufficient could accumulate to obstruct the catheter. DESIGN Elution of proteins from 102 explanted shunt catheters, with protein assay and electrophoresis of the eluate, and scanning electron microscopy (SEM) of the catheters. RESULTS The amount of protein elutable was extremely low, and significant protein, apart from a thin film, was not found on SEM. Qualitative analysis disclosed that most of the adsorbed protein was albumin. CONCLUSIONS Protein deposition on hydrocephalus catheters does not occur in sufficient quantities to cause catheter obstruction.