Microfluidic validation of diagnostic protein markers for spontaneous cerebrospinal fluid rhinorrhea.

Cerebrospinal fluid (CSF) rhinorrhea is a potentially dangerous condition identified by CSF leakage into the nasal cavity. This malady stands to benefit from rapid and noninvasive screening diagnostics to complement low-throughput imaging based methods currently in use. To address this gap, we demonstrate on-chip immunosubtraction to accelerate biomarker validation and immunoassay development for a putative CSF rhinorrhea diagnostic marker, transthyretin, by combining high-specificity immunoaffinity capture with subsequent polyacrylamide gel electrophoresis (PAGE). We demonstrate the on-chip assay using photopatterned polyacrylamide immunofilters. The filter consists of polymer with controlled pore-sizes to size-exclude (i.e., "subtract") large antibody-target immune complexes from downstream PAGE separation. A control PAGE separation is also performed for comparison without immunoaffinity capture (i.e., no antibody present). We compare on-chip immunosubtraction to Western blotting and ELISA to validate CSF rhinorrhea biomarkers from nasal surgery samples. For samples representative of spontaneous rhinorrhea, the 5 min on-chip assay achieved clinical specificity of 100%, compared to 50% for ELISA which required 6 h. On-chip immunosubtraction also generated results for clinical samples not assayable via ELISA due to matrix protein spurious signals. The pilot study suggests the capability of a rapid on-chip validation tool to expedite scrutiny of putative protein markers for new clinical assays.

Connective tissue disorders in patients with spontaneous intracranial hypotension.

OBJECTIVE: Spontaneous intracranial hypotension (SIH) is caused by spinal cerebrospinal fluid (CSF) leakage. An underlying connective tissue disorder has been hypothesized to cause dural weakness and predisposition to CSF leak. We conducted a case-controlled study to investigate the role of connective tissue disorders in SIH patients. METHODS: We recruited 55 consecutive SIH patients (38 F, 17 M; mean age, 40.8 ± 9.8 years) and 55 age- and sex-matched control individuals (mean age, 38.0 ± 8.9 years) for this study. The connective tissue disorders were evaluated by: (i) Beighton hypermobility scores and revised diagnostic criteria for benign joint hypermobility syndrome; (ii) skin and skeletal manifestations of Ehlers-Danlos syndrome (EDS); and (iii) skeletal features of Marfan syndrome. RESULTS: The frequencies of joint hypermobility according to Beighton scores >4/9 (SIH 23.6% vs controls 16.4%, P = 0.48) and revised benign joint hypermobility syndrome criteria (SIH 23.6% vs controls 34.5%, P = 0.29) did not differ between SIH patients and controls. Sixteen patients and 16 controls had one or more skin features of EDS (P = 1.0). Nine SIH patients (16.4%) demonstrated the skeletal features of Marfan syndrome; this frequency did not differ from that of the control group (9.1%; P = 0.262). Only dolichostenomelia (disproportionately long limbs) was more prominent in SIH patients than in controls (34.5% vs 9.1%; P = 0.002). CONCLUSION: Compared with Western studies, the frequencies of connective tissue disorders were higher in our SIH patients. However, these frequencies did not differ between SIH patients and control individuals, except for dolichostenomelia.

Beta-2 transferrin is detectable for 14 days whether refrigerated or stored at room temperature.

BACKGROUND: The effect of time and temperature on beta-2 transferrin stability in cerebrospinal fluid (CSF) is not well established. After collecting nasal CSF for testing, beta-2 transferrin has been found to be stable and detectable for 1 week, whether being refrigerated or stored at room temperature. The purpose of this study was to determine if beta-2 transferrin remained detectable longer than 1 week and whether refrigeration improved its detectability. METHODS: In patients undergoing therapeutic CSF diversion, 2-mL CSF samples were collected from 18 patients. The samples were divided and stored either at room temperature, or at 4°C, and tested for beta-2 transferrin at 7 and 14 days. CSF was collected from external ventricular drains (EVDs) (n = 15), lumbar drains (n = 2), and subdural drains (n = 1). RESULTS: Of the 18 CSF samples originally testing positive for beta-2 transferrin, none turned negative at 7 or 14 days, in both the refrigerated and room temperature groups (95% confidence interval [CI], 0% to 18.5%). CONCLUSION: Beta-2 transferrin remained detectable for 14 days in all CSF samples, regardless of being stored at 4°C or room temperature.

Cerebrospinal Fluid Infection Associated with Silver-Impregnated External Ventricular Drain Catheters.

OBJECTIVE: Silver-impregnated external ventricular drains (EVDs) have reduced the number of cases of ventriculitis. Risk factors for developing ventriculitis associated with plain EVD catheters have been well documented and incorporated into clinical practice, but risk factors in association with silver EVDs are unknown. Therefore, we sought to determine the risk factors for developing ventriculitis in patients with a silver EVD. METHODS: Prospectively collected data on consecutive patients undergoing insertion of a silver-impregnated EVD between October 2011 and September 2013 were analyzed. Cerebrospinal fluid (CSF) infection was defined as positive CSF microbiologic culture. Univariate and multivariate regression was performed to identify independent risk factors for CSF infection. RESULTS: There were 362 EVDs placed in 263 patients (142 women; median age 52 years; interquartile range, 40-62 years). There were 15 infections (6.4 per 1000 days of EVD drainage) recorded. Gram-negative organisms (8 of 15) were most commonly observed, although Staphylococcus was the most common genus (7 of 15; 46%) identified. Univariate analysis indicated that patients requiring EVD replacement (P < 0.0001), patients requiring bilateral EVDs (P < 0.0001), and patients with a CSF leak (P < 0.0001) were at increased risk of infection. Only need for EVD replacement remained significant on multivariate analysis (P < 0.0001, odds ratio = 15.9, confidence interval = 4.5-55.9). CONCLUSIONS: We identified an infection rate of 5.2% in this large contemporary series of patients undergoing silver EVD insertion according to a set protocol. These data suggest that targeting strategies to reduce Staphylococcus species is important, and increased vigilance for an increased incidence of gram-negative organisms is needed. Decreasing the need for EVD replacement is important in reducing infection rates further.

CSF proteome: a protein repository for potential biomarker identification.

Proteomic analysis is not limited to the analysis of serum or tissues. Synovial, peritoneal, pericardial and cerebrospinal fluid represent unique proteomes for disease diagnosis and prognosis. In particular, cerebrospinal fluid serves as a rich source of putative biomarkers that are not solely limited to neurologic disorders. Peptides, proteolytic fragments and antibodies are capable of crossing the blood-brain barrier, thus providing a repository of pathologic information. Proteomic technologies such as immunoblotting, isoelectric focusing, 2D gel electrophoresis and mass spectrometry have proven useful for deciphering this unique proteome. Cerebrospinal fluid proteins are generally less abundant than their corresponding serum counterparts, necessitating the development and use of sensitive analytical techniques. This review highlights some of the promising areas of cerebrospinal fluid proteomic research and their clinical applications.

New insights into the glucose oxidase stick test for cerebrospinal fluid rhinorrhoea.

Rhinorrhoea is a clinical sign of cerebrospinal fluid (CSF) leakage in patients with skull fracture, but can also be attributable to respiratory secretions or tears. Laboratory tests confirming the presence of CSF are not sufficiently rapid to support clinical decision making in the emergency department and may not be universally available. Detection of glucose in nasal discharge was traditionally used to diagnose CSF leak at the bedside, but has fallen into disuse as it has poor positive predictive value. We propose an algorithm to improve the diagnostic value of this test taking into consideration factors we have found to affect the glucose concentration of respiratory secretions. In patients at risk of CSF leak, nasal discharge is likely to contain CSF if glucose is present in the absence of visible blood, if blood glucose is <6 mmol x L(-1), and if there are no symptoms of upper respiratory tract infection.

A time-dependent peak of oxytocin exists in cerebrospinal fluid but not in plasma of humans.

Oxytocin (OT) and the oxytocin-neurophysin (OT-Np) were measured by RIA in samples of cerebrospinal fluid (CSF) obtained sequentially at 0600, 1200, 1800, and 2400 h from six patients in whom intrathecal catheters were temporarily placed for CSF rhinorrhea. The highest levels of OT in CSF were found at 1200 h. An analysis of variance of sequential measures of the concentration of OT in samples of CSF obtained every 6 h over a 30-h period showed the mean levels (+/- SEM) of OT at 1200 h, 6.41 +/- 1.13 microU/ml and 5.06 +/- 0.58 microU/ml, to be significantly higher (p less than 0.05) than mean levels of OT at 0600 h, 2.50 +/- 0.65 microU/ml; 1800 h, 2.63 +/- 0.61 microU/ml and 2.64 +/- 1.21 microU/ml; and 2400 h, 2.86 +/- 1.13 microU/ml. The levels of OT-Np in CSF did not show a similar peak. In three of the patients simultaneous samples of blood were obtained for measurement of the same peptides, but no corresponding peak of OT or its Np was found in plasma of these three patients. The level of OT in CSF at all times was also significantly higher (p less than 0.05) than the level of OT in plasma of these three patients. Levels of OT and OT-Np were measured by RIA of samples of plasma obtained hourly for a 24-h period from six healthy men and six healthy women. No diurnal variation of OT or its Np in the plasma of men or women was found. This pattern of OT in the CSF of humans is similar to the pattern of OT in the CSF of the Rhesus monkey, but in contrast to the lack of a clearly defined peak of OT in the CSF of the cat or the rat. These observations in humans reinforce the differences among species of the secretion of OT in the CSF.

Predictive values of beta-trace protein (prostaglandin D synthase) by use of laser-nephelometry assay for the identification of cerebrospinal fluid.

OBJECTIVE: beta-Trace protein (beta-TP) is an immunological marker for the detection of cerebrospinal fluid traces. The aim of the study was to evaluate the predictive values of a new research assay for beta-TP. METHODS: A total of 154 specimens from patients with otorrhea or rhinorrhea were investigated for cerebrospinal fluid (CSF) by use of a laser-nephelometric assay for beta-TP. Samples were obtained between January 1994 and November 2000. A sample was reported to be positive for CSF when the beta-TP concentration was more than 6 mg/L. Case evaluations were performed retrospectively and tabulated for indication, clinical course, additional investigations, surgical procedure, and follow-up. RESULTS: beta-TP was detected in 16 specimens; 138 samples were negative for beta-TP with a value less than 3 mg/L. One sample was suggestive of CSF traces at 4.6 mg/L. In correlation with the clinical course, the intraoperative findings, intraoperative visualization with sodium fluorescein, high-resolution computed tomography of the paranasal sinuses or the petrous bone, computed tomographic cisternography, magnetic resonance imaging, and radionuclide cisternography, there was no false-positive result. On four occasions, false-negative results occurred, with an overall accuracy of 0.974. The beta-TP test had a negative predictive value of 0.971 and a positive predictive value of 1. CONCLUSION: Analysis of betas-TP via the nephelometric assay is a valuable and reliable test in cranial base surgery for the identification of CSF.

Cerebrospinal fluid rhinorrhoea and Haemophilus influenzae meningitis 37 years after a head injury.

Bacterial meningitis secondary to CSF rhinorrhoea is well recognised. We present a case of meningitis due to Haemophilus influenzae type b associated with a CSF fistula. The patient was 40 years of age at presentation, having sustained a head injury when 3-years-old. He had suffered intermittent rhinorrhoea for 18 months before admission. Delays between head injury and meningitis are well recognised, but our case may represent the longest recorded interval. H. influenzae meningitis in adults is discussed.

Electrophoretic study of the cerebrospinal fluid proteins of a patient with multiple sclerosis and recurrent meningitis (with rhinorrhea).

Case report on a 33 year old multiple sclerosis patient showing recurrent meningitis with an episode of rhinorrhea. The emphasis was put on the protein electrophoresis patterns.

A new method for the identification of cerebrospinal fluid leakage.

Apart from the use of anamnestic data the most common techniques for the diagnosis of CSF leakage have included X-ray studies, and chemical analysis of glucose, protein, and electrolytes of the fluid obtained from the nose or ear, intrathecal staining, and radioactive cisternography. These studies, although useful, have not always succeeded in demonstrating the CSF leakage, especially when the leak is delayed, small, or contaminated. A new immunochemical technique for the identification of the CSF leakage is described. It is based on demonstration of an extra band of transferrin located in the beta 2-fraction of protein electrophoresis of CSF. This beta 2-transferrin is pathognomonic for liquor and could not be demonstrated in serum, nasal secretions, saliva, tears, or peri- and endolymph. After routine protein electrophoresis on cellulose acetate membranes, the transferrins are identified by application of anti-transferrin on beta-regions. Stained precipitates in both beta-regions demonstrate clearly the presence of CSF. Compared with other methods, the new technique offers many advantages. The amount of sample needed for the procedure is small, moderate contamination does not invalidate it, it makes it possible to localize the leak by differential suction, and it is absolutely safe for the patient.

Spontaneous cerebrospinal fluid rhinorrhea as a presenting symptom of aqueductal stenosis--case report.

A 30-year-old male patient presented with chronic spontaneous cerebrospinal fluid (CSF) rhinorrhea. He had sustained a mild head injury in childhood. Magnetic resonance imaging of the brain showed aqueductal stenosis associated with moderate supratentorial hydrocephalus, and erosion of the cribriform plate. Following insertion of ventriculoperitoneal shunt, the CSF rhinorrhea completely ceased and no direct repair of the CSF fistula was necessary. Long-standing spontaneous CSF rhinorrhea indicates the possibility of concurrent intracranial pathology, such as aqueductal stenosis.

[The value of beta-2 transferrin analysis and MR cisternography for the diagnosis of cerebrospinal fluid fistulas]. / Intérêt de la recherche de beta2 transferrine et de la MR cisternographie pour le diagnostic des fistules de liquide céphalorachidien.

The diagnosis of cerebrospinal fluid (CSF) fistula may require invasive techniques. Detection of CSF and perilymph-specific beta 2 transferrin and MR cisternography which greatly enhances the CSF signal are sensitive and noninvasive techniques which allowed a precise diagnosis in seven patients with suspected CSF fistula. We review the different diagnostic techniques used for CSF fistula, beta 2 transferrin analysis and MR cisternography appear to provide accurate and noninvasive methods for investigating CSF fistulae. They should replace invasive techniques such as CT cisternography.

[A case of spontaneous rhinorrhea detected by SPAMM-tagging method].

This is the first report of rhinorrhea detected by special modulation of the magnetization (SPAMM)-tagging method. A 54-year-old male was admitted to our hospital for treatment of right rhinorrhea which had continued for 2 months and a half. The CSF leakage was induced by specific head position and the volume was more than 50 ml a day. Metrizamide CT cisternography revealed the contrast medium in the lateral extension of the right sphenoid sinus. MRI demonstrated liquorrhea with abnormal intensity of the sinus. 3D-CT revealed bony defects at the temporal base. Operation revealed herniated brain through the same bony defect of the temporal base into the extended sphenoid sinus. Post-operative diagnosis was Morley's rhinorrhea. The leakage point was closed and patched with the femoral fascia. Preoperative SPAMM-tagging image with cine MRI was useful to identify the responsible leakage point as a disorder of lattice tags. This method is a kind of flowmetry with MRI and is very effective because it can detect non-invasively slight CSF motion into the sinuses. It can also detect the direction and rough flow volume of rhinorrhea, so may also predict the risk of meningitis. The disadvantages were also discussed.

[Cerebrospinal fluid transfer of cefotiam (CTM). Its relationship to CT].

Drip intravenous infusion of cefotiam (CTM) was made on patients who underwent cerebrospinal fluid (CSF) drainage and study was made on CSF transfer of CTM and at the same time on the relationship between CSF transfer of iodine contrast medium and CT scan findings. This study was made on 11 cases of cisternal drainage and 8 cases of ventricular drainage. Cisternal drainage cases were all postoperative cases of ruptured cerebral aneurysm. Cases of ventricular drainage included 4 postoperative cases of ruptured cerebral aneurysm, 1 case of CSF rhinorrhea, 2 cases of brain tumor, and 1 case of ventricular hemorrhage. Drip intravenous infusion of 1.0 g of CTM was made in one hour and at given periods thereafter CSF was collected and measured. CTM transferred to the CSF in cistern at a comparatively high concentration (16.3-0.7 microgram/ml). Hardly any transfer of CTM to the CSF in ventricle was seen in one case of cerebral aneurysm, CSF rhinorrhea, and brain tumor, but transfer was observed in one case of cerebral aneurysm, one case of brain tumor, and case of ventricular hemorrhage. Transfer of iodine contrast medium showed the positive correlation to the transfer of CTM. In cases of brain tumor and ventricular hemorrhage with transfer of CTM with ventricular drainage, enhancement effect of the ventricular wall by the contrast medium could be observed by CT scan. From the foregoing, the following results were obtained. There was good transfer of CTM to the CSF in cistern in postoperative cases of ruptured aneurysm. CTM did not transfer to CSF in the normal ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)

[Diagnosis and surgical treatment of liquorrhea]. / Diagnostikk og kirurgisk behandling av likvoré.

BACKGROUND: Liquorrhea caused by cerebrospinal fluid fistula may lead to meningitis and should be treated surgically. The diagnostic approach and the surgery may be difficult and improvements are needed. MATERIAL AND METHODS: We present two cases of liquorrhea. A review of the diagnostic and surgical methods is given. RESULTS: In both cases the diagnosis was confirmed by detection of beta-trace protein. In one case the surgery was complicated. This patient had a cerebrospinal fluid fistula from the sphenoid sinus. In a third operation, performed with sodium fluorescein dying, the fistula was closed. INTERPRETATION: Rhinorrhea and meningitis are unspecific signs of a possible cerebrospinal fluid fistula. Beta-trace protein is recommended as a diagnostic marker, because this protein has the highest positive predictive value for the presence of cerebrospinal fluid. High-resolution CT scans are the most helpful imaging technique for determining the leakage site, but this is not conclusive in all cases. Using sodium fluorescein may give better outcomes of surgical interventions.