Guidelines for uniform reporting of body fluid biomarker studies in neurologic disorders.

OBJECTIVE: The aim of these guidelines is to make the process of reporting body fluid biomarker studies in neurologic disorders more uniform and transparent, in line with existing standards for reporting research in other biomedical areas. Although biomarkers have been around for decades, there are concerns over the high attrition rate of promising candidate biomarkers at later phases of development. METHODS: BioMS-eu consortium, a collaborative network working toward improving the quality of biomarker research in neurologic disorders, discussed the merits of standardizing the reporting of body fluid biomarker research. A checklist of items integrating the results of other published guidances, literature, conferences, regulatory opinion, and personal expertise was created to ultimately form a structured summary guidance incorporating the key features. RESULTS: The summary guidance is comprised of a 10-point uniform reporting format ranging from introduction, materials and methods, through to results and discussion. Each item is discussed in detail in the guidance report. CONCLUSIONS: To enhance the future development of body fluid biomarkers, it will be important to standardize the reporting of studies. This guideline by the BioMS-eu consortium is aimed at setting a standard for the reporting of future body fluid biomarker research studies in neurologic disorders. We anticipate that following these guidelines will help to accelerate the selection of biomarkers for clinical development.

Association of cerebral microbleeds in acute ischemic stroke with high serum levels of vascular endothelial growth factor.

OBJECTIVE: To determine whether vascular endothelial growth factor (VEGF) levels are associated with the presence of cerebral microbleeds (CMBs) in patients after acute ischemic stroke. DESIGN: A cross-sectional study that used blood samples obtained within 24 hours of symptom onset from patients who experienced acute stroke to measure VEGF levels by enzyme immunoassay. A validated CMB rating scale was used to analyze acutely acquired magnetic resonance images, with the rater blind to clinical details and VEGF levels. SETTING: Accident and Emergency Department at University College Hospital, London, England. PATIENTS: Twenty patients who experienced acute ischemic stroke. MAIN OUTCOME MEASURES: Presence of CMBs and serum level of VEGF. RESULTS: Five of the 20 patients with acute ischemic stroke (25%) had CMBs. The median VEGF level in the CMB group was significantly higher than that in the group without CMBs (P = .003). CONCLUSION: An increase in vascular permeability secondary to a raised VEGF level may have a role in the genesis of CMBs in patients with acute ischemic stroke.

Value of measuring serum vascular endothelial growth factor levels in diagnosing acute ischemic stroke.

BACKGROUND AND PURPOSE: It has previously been reported that serum levels of vascular endothelial growth factor are raised after acute ischemic stroke compared to healthy controls. The aim of this prospective study was to ascertain whether serum vascular endothelial growth factor measurements could be used to distinguish between acute ischemic stroke and common stroke mimics in the emergency room. METHODS: Blood samples were taken on arrival to hospital and daily for six-days, in 44 patients with suspected ischemic stroke (29 acute infarcts and 15 stroke mimics), arriving within 24 h of symptom onset. Vascular endothelial growth factor levels were measured by enzyme-linked immunoassay. The neurological deficit was recorded daily using the National Institute of Health Stroke Scale. Evaluation of infarct volumes was based on diffusion-weighted magnetic resonance imaging. RESULTS: Serum vascular endothelial growth factor levels were significantly raised in acute ischemic stroke patients on the day of symptom onset and at all other time points, compared to healthy controls (P < 0·01). The sensitivity and specificity of vascular endothelial growth factor for diagnosing acute ischemic stroke on admission to hospital were only 69% and 73%, respectively. Vascular endothelial growth factor levels were also elevated in four out of 15 stroke mimics, including three patients presenting with postictal paresis. CONCLUSIONS: Vascular endothelial growth factor has limited clinical utility in the diagnosis of acute ischemic stroke in the emergency room because levels are also raised in common stroke mimics. Further studies are required to establish the mechanism of vascular endothelial growth factor elevation in postictal paresis.

Increase of uric acid and purine compounds in biological fluids of multiple sclerosis patients.

OBJECTIVES: In this study, the concentrations of uric acid, purine profile and creatinine in samples of cerebrospinal fluid and serum of multiple sclerosis (MS) patients were measured by HPLC and compared with corresponding values recorded in patients without MS (cerebrospinal fluid) and healthy subjects (serum). DESIGN AND METHODS: All samples were deproteinized with ultrafiltration (which ensures minimal sample manipulation and efficient protein removal) and then assayed for the synchronous HPLC separation of uric acid, hypoxanthine, xanthine, inosine, adenosine, guanosine and creatinine. RESULTS: The values of all compounds assayed were significantly higher in both biological fluids of MS patients with respect to values measured in controls. In particular, serum hypoxanthine, xanthine, uric acid and sum of oxypurines were, respectively, 3.17, 3.11, 1.23 and 1.27-fold higher in these patients than corresponding values recorded in controls (p<0.001). CONCLUSIONS: Differently from what previously reported, we here demonstrate that all purine compounds, including uric acid, are elevated in biological fluids of MS patients. Reinforced by the trend observed for creatinine, this corroborates the notion of sustained purine catabolism, possibly due to imbalance in ATP homeostasis, under these pathological conditions. These results cast doubt on the hypothesis that uric acid is depleted in MS because of increased oxidative stress, rather suggesting that this disease causes a generalized increase in purine catabolism. As observed in other pathological states, uric acid, purine compounds and creatinine, can be considered markers of metabolic energy imbalance rather than of reactive oxygen species, even in MS.

Molecular dissection of Alzheimer's disease neuropathology by depletion of serum amyloid P component.

New therapeutic approaches in Alzheimer's disease are urgently needed. The normal plasma protein, serum amyloid P component (SAP), is always present in cerebrospinal fluid (CSF) and in the pathognomonic lesions of Alzheimer's disease, cerebrovascular and intracerebral Abeta amyloid plaques and neurofibrillary tangles, as a result of its binding to amyloid fibrils and to paired helical filaments, respectively. SAP itself may also be directly neurocytotoxic. Here, in this unique study in Alzheimer's disease of the bis(d-proline) compound, (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), we observed depletion of circulating SAP and also remarkable, almost complete, disappearance of SAP from the CSF. We demonstrate that SAP depletion in vivo is caused by CPHPC cross-linking pairs of SAP molecules in solution to form complexes that are immediately cleared from the plasma. We have also solved the structure of SAP complexed with phosphothreonine, its likely ligand on hyperphosphorylated tau protein. These results support further clinical study of SAP depletion in Alzheimer's disease and potentially other neurodegenerative diseases.

Longitudinal one-year study of levels and stoichiometry of neurofilament heavy and light chain concentrations in CSF in patients with multiple system atrophy.

BACKGROUND: Two cerebrospinal fluid (CSF) biomarkers specific for neurodegeneration have recently emerged - the neurofilament light (NfL, 68 kDa) and heavy (NfH, 190-210 kDa) chains. This study investigated whether the CSF NfH and NfL levels or their stoichiometric relationship changed over time in a neuroprotective treatment trial. METHODS: Serial CSF samples (n=95) from 42 patients with multiple system atrophy (MSA), half randomized to treatment with recombinant human growth hormone (r-hGH) and the other half to placebo, were collected at baseline, 6 and 12 months. The concentration of CSF NfL and NfH was determined using standard ELISAs. RESULTS: There was no consistent change in the levels of either protein over the 12 month period, or between treatment with active r-hGH versus placebo. The molar stoichiometry of CSF NfL:NfH was 4:1 (R=0.37, p=0.0002) and increased following treatment with r-hGH (p=0.03). CONCLUSION: These results indicate that CSF levels of both NfL and NfH on their own are not useful markers of disease progression in MSA, at least over a 12-month period. Future work is needed to elucidate whether the CSF stoichiometry and dynamics of Nf subunits in individual patients are a feature of the underlying pathology and of diagnostic or prognostic value.

Criteria for a clinically informative serum biomarker in acute ischaemic stroke: a review of S100B.

BACKGROUND AND PURPOSE: Serum S100B has been widely studied as a biomarker in acute ischaemic stroke. The main objective of this review was to appraise the published literature on S100B and determine its clinical applicability. METHODS: Medline was searched to identify studies on S100B (or S-100B) in acute ischaemic stroke. The authors have proposed the criteria for a clinically informative serum biomarker for acute ischaemic stroke, and relevant articles relating to these criteria were then selected. RESULTS: Studies have shown that S100B has a low specificity for acute ischaemic stroke because of its tendency to be raised from extracranial sources. Data regarding S100B kinetics compiled from 6 longitudinal studies show that serum levels are not raised immediately following acute ischaemic stroke and peak 3 days after symptom onset. However, serum S100B levels correlate well with infarct volume and are higher in stroke patients at risk of malignant infarction or haemorrhagic transformation after thrombolysis. In addition, serum S100B levels correlate well with functional outcome. CONCLUSION: The evidence suggests that S100B is not a valuable biomarker for diagnosing acute ischaemic stroke. Instead, it may have a more promising role in non-specialist hospitals, as an additional tool for identifying patients at increased risk of specific early neurological complications after stroke and as a surrogate marker of cerebral damage and functional outcome, particularly in a research setting.

Phosphorylation and compactness of neurofilaments in multiple sclerosis: indicators of axonal pathology.

AIMS: Axonal pathology extends to the axonal cytoarchitecture leaving its signature on axoskeletal proteins. This study investigated whether neurofilament (NfH) phosphorylation would relate to the dynamics of axonal pathology in multiple sclerosis (MS). METHODS: NfH phosphoforms (SMI32, SMI34, SMI35) were quantified by ELISA from microdissected samples of control and MS brain and spinal cord. Individual axons were analysed by electron microscopy, densitometrically and morphologically in adjacent tissue sections. Experiments were carried out pre- and post enzymatic dephosphorylation. RESULTS: In control tissue a rostro-caudal gradient of NfH indicated an increase in axonal density from the brain gray matter towards the spinal cord. The highest levels of phosphorylated and hyperphosphorylated NfH were found in acute lesions of brain and spinal cord, in contrast to chronic lesions where levels were lower than in white matter, consistent with axonal loss. Dephosphorylated NfH was higher, but less densly packed in MS white matter axons compared to control tissue. CONCLUSIONS: The findings suggest that a less organised/compact axoskeleton or impaired axonal transport may represent an early sign of axonal pathology within the normal appearing white matter in MS. Subsequently a proportional increase of dephosphorylated NfH, aberrant phosphorylation and/or aggregation may occur whilst the protein is transported through the white matter towards the MS plaque, where hyperphosphorylated NfH dominates.

Discrepancies between qualitative and quantitative analysis of cerebrospinal fluid IgG: densitometric comparison of nephelometry-silver versus immuno-enzymes.

BACKGROUND: Detection of local synthesis of IgG within the central nervous system is important for the diagnosis of brain inflammatory diseases such as multiple sclerosis. This is typically done by comparing the amounts of IgG in serum and parallel cerebrospinal fluid (CSF). Although there have been well-described problems with qualitative versus quantitative measurements of abnormal IgG, such as in myeloma paraproteins, similar difficulties are also found with CSF IgG. METHODS: Traditional quantitative analysis of IgG by rate nephelometry was followed by separation of the IgG using isoelectric focusing and then either silver stain or immunofixation. Finally, quantitative analysis was performed by scanning densitometry using public domain software downloaded from the National Institutes of Health. RESULTS: We report here the major discrepancies that can occur with CSF IgG between the silver stain versus the IgG stain. CONCLUSIONS: We concur with the earlier recommendation that qualitative separation followed by densitometric estimation of enzyme-linked immunofixation is also more useful than simple quantitative nephelometric analysis followed by silver staining in the detection of local synthesis of IgG, analogous to the earlier work on paraproteins.

Imaging cadavers: cold FLAIR and noninvasive brain thermometry using CSF diffusion.

There is increasing interest in imaging cadavers for noninvasive autopsies for research purposes. However, the temperature is well below that of in vivo imaging, and a variety of interesting 'cold brain' effects are observed. At lower temperatures conventional FLAIR sequences no longer produce dark cerebrospinal fluid (CSF); T(1) is reduced from about 4.0 sec in vivo to 1.7 sec at 1 degrees C. The diffusion coefficient (DC) of CSF is much reduced (from 3.1 10(-9) m(2)s(-1) in vivo to 1.1 at 1 degrees C). DC values therefore provide a noninvasive thermometer to measure brain core temperature to within 1.0 degrees C. In three cadavers DC values were 1.1-1.5 10(-9) m(2)s(-1), indicating brain core temperatures of 1-10 degrees C, consistent with external thermocouple measurements. An improved inversion time (TI(0)) can then be found for FLAIR. At 10 degrees C this Cold FLAIR sequence (TI(0) = 1.5 sec) gave black CSF. Expressions for CSF DC and T(1) as a function of temperature were produced. A measurement of CSF DC could be converted directly to temperature and the required TI(0) found. In vitro values of CSF DC were about 1% lower than that of water. Thus, FLAIR imaging can be optimized for cadaveric brains at low and unknown temperatures, thereby improving value for autopsy purposes and facilitating comparisons with in vivo imaging.

Marathon related death due to brainstem herniation in rehydration-related hyponatraemia: a case report.

INTRODUCTION: Identifying marathon runners at risk of neurological deterioration at the end of the race (within a large cohort complaining of exhaustion, dehydration, nausea, headache, dizziness, etc.) is challenging. Here we report a case of rehydration-related hyponatraemia with ensuing brain herniation. CASE PRESENTATION: We report the death of runner in his 30's who collapsed in the recovery area following a marathon. Following rehydration he developed a respiratory arrest in the emergency room. He was found to be hyponatraemic (130 mM). A CT brain scan showed severe hydrocephalus and brain stem herniation. Despite emergency insertion of an extraventricular drain, he was tested for brainstem death the following morning. Funduscopy demonstrated an acute-on-chronic papilledema; CSF spectrophotometry did not reveal any trace of oxyhemoglobin or bilirubin, but ferritin levels were considerably raised (530 ng/mL, upper reference value 12 ng/mL), consistent with a previous bleed. Retrospectively it emerged that the patient had suffered from a thunderclap headache some months earlier. Subsequently he developed morning headaches and nausea. This suggests that he may have suffered from a subarachnoid haemorrhage complicated by secondary hydrocephalus. This would explain why in this case the relatively mild rehydration-related hyponatremia may have caused brain swelling sufficient for herniation. CONCLUSION: Given the frequency of hyponatraemia in marathon runners (serum Na <135 mM in about 13%), and the non-specific symptoms, we discuss how a simple screening test such as funduscopy may help to identify those who require urgent neuroimaging.

Early identification of secondary brain damage in subarachnoid hemorrhage: a role for glial fibrillary acidic protein.

Secondary ischaemic deficit adversely affects outcome in patients with subarachnoid hemorrhage (SAH). Astrocytes are vulnerable to ischemia, releasing glial fibrillary acidic protein (GFAP) when challenged. In this study, we followed nine patients with SAH who underwent extra-ventricular drainage for the management of secondary hydrocephalus. Cerebrospinal fluid (CSF) was collected daily for up to 14 days. CSF GFAP was quantified using a standard ELISA. In the patients, we found that the CSF GFAP values were pathologically elevated in 83/89 (93%) of the CSF samples. The levels were highest on day 1 (median = 47.64 ng/mL) and decreased to 11.19 ng/mL on day 3, leveling out at approximately 1 ng/mL after 10 days. In non-survivors, a secondary rise of GFAP levels became significant during the high-risk period for vasospasm, with median levels of 21.76 ng/mL compared to 2.62 ng/mL in the survivors (p = 0.037) on day 6. This study suggests that CSF GFAP levels are of prognostic value in SAH. Additionally, the difference in the slope of GFAP levels between survivors (rapid wash-out) and non-survivors (secondary peaks) may allow difierentiation between primary brain injury from secondary brain damage due to delayed cerebral ischaemia.

Spectrophotometry for cerebrospinal fluid pigment analysis.

The use of spectrophotometry for the analysis of the cerebrospinal fluid (CSF) is reviewed. The clinically relevant CSF pigments--oxyhemoglobin and bilirubin--are introduced and discussed with regard to clinical differential diagnosis and potentially confounding variables (the four T's: traumatic tap, timing, total protein, and total bilirubin). The practical laboratory aspects of spectrophotometry and automated techniques are presented in the context of analytical and clinical specificity and sensitivity. The perceptual limitations of human color vision are highlighted and the use of visual assessment of the CSF is discouraged in light of recent evidence from a national audit in the United Kingdom. Finally, future perspectives including the need for longitudinal CSF profiling and routine spectrophotometric calibration are outlined.

Diagnosis of congenital disorders of glycosylation type-I using protein chip technology.

A method for the diagnosis of the congenital disorders of glycosylation type I (CDG-I) by SELDI-TOF-MS of serum transferrin immunocaptured on protein chip arrays is described. The underglycosylation of glycoproteins in CDG-I produces glycoforms of transferrin with masses lower than that of the normal fully glycosylated transferrin. Immobilisation of antitransferrin antibodies on reactive-surface protein chip arrays (RS100) selectively enriched transferrin by at least 100-fold and allowed the detection of patterns of transferrin glycoforms by SELDI-TOF-MS using approximately 0.3 microL of serum/plasma. Abnormal patterns of immunocaptured transferrin were detected in patients with known defects in glycosylation (CDG-Ia, CDG-Ib, CDG-Ic, CDG-If and CDG-Ih) and in patients in whom the basic defect has not yet been identified (CDG-Ix). The correction of the N-glycosylation defect in a patient with CDG-Ib after mannose therapy was readily detected. A patient who had an abnormal transferrin profile by IEF but a normal profile by SELDI-TOF-MS analysis was shown to have an amino acid polymorphism by sequencing transferrin by quadrupole-TOF MS. Complete agreement was obtained between analysis of immunocaptured transferrin by SELDI-TOF-MS and the IEF profile of transferrin, the clinical severity of the disease and the levels of aspartylglucosaminidase activity (a surrogate marker for the diagnosis of CDG-I). SELDI-TOF-MS of transferrin immunocaptured on protein chip arrays is a highly sensitive diagnostic method for CDG-I, which could be fully automated using microtitre plates and robotics.

A screening assay to detect antigen-specific antibodies within cerebrospinal fluid.

Identification of the aetiology of central nervous system infections requires the detection of either the organism or a microbe-specific immune response within the brain or cerebrospinal fluid. We describe a screening assay to detect herpes simplex virus, varicella zoster virus, cytomegalovirus, measles and Toxoplasma gondii specific antibodies in cerebrospinal fluid. Antigen-specific immunoblotting of oligoclonal IgG and IgM was used to confirm the presence of antibody. Of 51 consecutive cerebrospinal fluid samples received by the laboratory from patients with suspected central nervous system infection 18 (35%) were screen positive for one or more antigen. In only 7 of these were antigen-specific oligoclonal IgG or IgM bands confirmed. The assay provides a simple, cheap assay to screen for microbial-specific antibody in the cerebrospinal fluid samples of patients with suspected neurological infections.

Measurement of high affinity antibodies on antigen-immunoblots.

We describe a semi-quantitative method for measuring the relative affinity of antigen-specific oligoclonal IgG bands separated by isoelectric focusing followed by blotting onto antigen-coated membrane and incubation with sodium thiocyanate. When the developed blot is digitised in greyscale, densitograms can be made and peak areas calculated using ImageJ freeware. By expressing peak area as a percentage of the total area under the curve we have shown that there is a statistically significant rise in percentage of peak area for a given band which persists with increasing molarities of sodium thiocyanate.

Recommended standard of cerebrospinal fluid analysis in the diagnosis of multiple sclerosis: a consensus statement.

New criteria for the diagnosis of multiple sclerosis (MS) were published as the result of an internationally formed committee. To increase the specificity of diagnosis and to minimize the number of false diagnoses, the committee recommended the use of both clinical and paraclinical criteria, the latter involving information obtained from magnetic resonance imaging, evoked potentials, and cerebrospinal fluid (CSF) analysis. Although rigorous magnetic resonance imaging requirements were provided, the "new criteria paper" fell short in terms of guidelines as to how the CSF analysis should be performed and simply equated the IgG index with isoelectric focusing, without any justification. The spectrum of parameters analyzed and methods for CSF analysis differ worldwide and often yield variable results in terms of sensitivity, specificity, accuracy, and reliability, with no decided "optimal" CSF test for the diagnosis of MS. To address this question specifically, an international panel of experts in MS and CSF diagnostic techniques was convened and the result was this article, representing a consensus of all the participants. These recommendations for establishing a standard for the evaluation of CSF in patients suspected of having MS should greatly complement the new criteria in ensuring that a correct diagnosis of MS is being made.

Why human color vision cannot reliably detect cerebrospinal fluid xanthochromia.

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. CONCLUSIONS: We strongly recommend relying on spectrophotometry as the analytical method of choice.