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.

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.

Beta-trace protein in ascites and pleural effusions: limits of CSF leakage detection.

Rhino- and/or otoliquorrhea can be diagnosed by detecting beta-trace protein (ß-TP) in nasal or ear secretions, as ß-TP is found in high concentrations in cerebrospinal fluid (CSF) but not in serum. CSF fistulae following trauma or surgery can also occur at other anatomical sites, resulting in CSF leakage into the thoracic and abdominal cavities. By analogy, determination of ß-TP has also been used to diagnose CSF admixture in pleural effusions and ascites. However, no systematic study has yet evaluated the concentrations of ß-TP in such fluids in the absence of CSF. To determine the validity of ß-TP determination as a marker for the presence of CSF, we investigated ß-TP concentrations in pleural effusions and ascites without CSF admixture. Patients from whom samples of ascites or pleural effusion and a paired plasma sample were available were investigated. One hundred sixty-four patients were prospectively recruited. ß-TP concentrations were determined by nephelometry. Mass spectrometric proteome analysis confirmed the presence of ß-TP in the samples. Median ß-TP concentrations detected in ascites and pleural effusions (range, 0.014-26.5 mg/L, median 2.29 mg/L) exceeded the corresponding plasma concentrations 2.6-fold. According to cutoffs published to diagnose rhino- and otoliquorrhea, between 6.1% and 95.7% of the specimens would have been erroneously rated CSF-positive. Protein analysis confirmed the presence of ß-TP in pleural effusion and ascites. Ascites and pleural effusion contain high concentrations of ß-TP that exceed the levels in corresponding plasma. Therefore, ß-TP is not a specific marker for the presence of CSF in these fluids.

Preliminary study on the stability of beta-2 transferrin in extracorporeal cerebrospinal fluid.

OBJECTIVE: Detection of beta-2 transferrin in rhinorrhea fluid is a sensitive and specific method for the diagnosis of a cerebrospinal fluid (CSF) leak. Patients may be asked to collect this fluid at home to obtain an adequate volume for detection, and thus the age and storage conditions of these specimens may be variable upon analysis. The purpose of this study is to understand how age, storage temperature, and exposure to mucus affect the ability to detect beta-2 transferrin in CSF. STUDY DESIGN: Case series with planned data collection. SETTING: Tertiary care university hospital. SUBJECTS AND METHODS: This study consists of 6 patients undergoing endoscopic CSF leak repair. CSF was collected directly from a lumbar drain (n = 4) or from nasal drainage (n = 2). Specimens were stored at 4°C (n = 3) or room temperature (n = 3). Samples were tested for the presence of beta-2 transferrin for up to 7 days using standard immunofixation electrophoresis techniques. RESULTS: Beta-2 transferrin was detected in all specimens through day 7 regardless of storage temperature or collection site (95% exact binomial confidence interval of 0%-46%). CONCLUSIONS: Beta-2 transferrin remains detectable in extracorporeal CSF for up to 7 days regardless of storage at room temperature or exposure to nasal mucus. Negative detection in patient specimens up to a week old is therefore not likely to be caused by protein degradation.

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.

Rhinorrhea not responding to nasal corticosteroids.

A woman with multiple illnesses including allergic rhinitis presented for a follow-up visit at our clinic with constant rhinorrhea for 2 weeks despite regular use of nasal corticosteroids. Two weeks earlier, after alcohol drinking and doubling some of her medications for missed doses, she fell on her face. The Emergency Department records documented headache, bradycardia, hypotension, dehydration, and right infraorbital swelling. She was admitted for hydration and observation, and was discharged after two days without radiologic evaluation of the head. At our clinic, physical examination revealed pale turbinates bilaterally and clear watery discharge from the right nostril. Cerebrospinal fluid (CSF) rhinorrhea was suspected, but glucose testing was not available at our clinic. The patient was immediately admitted into the hospital. A beta-2-transferrin test confirmed CSF from the right nostril. High resolution sinus CT revealed fluid in the right sphenoid sinus, a large cyst in the left maxillary sinus, a cribriform plate dehiscence on the right side, and fluid collection adjacent to the middle turbinate. A lumbar drain was placed to release the pressure and antibiotic prophylaxis was started. Nasal endoscopy revealed CSF leak from the cribriform plate with bone dehiscence and a dural tear. A graft from nasal septal cartilage and temporalis fascia was applied using Tisseal fibrin glue. The persistent rhinorrhea resolved and on follow-up visits, the patient remained asymptomatic. Thinking of CSF rhinorrhea in the differential diagnosis of rhinitis would lead to early diagnosis and prevention of serious medical complications and potential legal liabilities.

Separation of beta2-transferrin by denaturing gel electrophoresis to detect cerebrospinal fluid in ear and nasal fluids.

BACKGROUND: Cerebrospinal fluid (CSF) leakage is a critical condition with a substantial risk of meningitis. We investigated the use of transferrin isoform analysis as a diagnostic marker for detection of CSF leakage in fluid samples. METHODS: We analyzed 241 samples from patients with CSF leakage, most commonly presenting as otorrhea or rhinorrhea, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent Western blotting and immunostaining for transferrin. Tears, saliva, nasal fluid, and ear secretions (20 samples each) were analyzed in parallel, and normal human serum served as a control in each experiment. We compared the minimum volume of added CSF that could be detected in secretions by our assay with the minimum volume detected by the prostaglandin-D synthase (beta-trace) test. CSF was admixed with blood in different proportions to determine the influence of blood contamination on the transferrin pattern. RESULTS: In all CSF samples, beta1- and beta2-transferrin were present in nearly equal amounts. In tears and ear secretions, beta2-transferrin migrated in the gel in the same manner as in CSF, but its concentration was noticeably lower than that of beta1-transferrin, a difference that allowed a clear distinction from the transferrin pattern of CSF. In saliva, both transferrin isoforms were also present but could be distinguished from those of other fluids by electrophoretic migration pattern rather than relative concentrations. With the beta-trace test, a minimum of 5 microL of CSF was needed for detection, whereas our beta2-transferrin assay yielded a signal of comparable intensity with a minimum of 2 microL of CSF. CONCLUSION: Analysis of the transferrin microheterogeneity pattern by SDS-PAGE for the identification of CSF leakage is a highly sensitive and specific method that merits consideration as a routine technique.

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.

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.

Rapid method for beta2-transferrin in cerebrospinal fluid leakage using an automated immunofixation electrophoresis system.

BACKGROUND: Beta(2)-transferrin (beta-2 trf) is a desialated isoform of transferrin found only in cerebrospinal fluid (CSF), ocular fluids, and perilymph. In aural, nasal, and wound drainages, this protein is an important marker of CSF leakage. Immunofixation electrophoresis (IFE) on agarose gels is a widely accepted qualitative technique for detection of small amounts of beta-2 trf, but disadvantages include lengthy transfer immunoblotting techniques or the requirement of at least 2 mL of sample. METHODS: Using eight applications of unconcentrated sample on high-resolution agarose gels with an automated electrophoresis system (Helena SPIFE 3000), we developed a rapid method for beta-2 trf. Evaluation studies included reproducibility of migration distance (mm), limit of detection, specificity, and concordance of results compared with those reported by a reference laboratory. Neuraminidase-treated serum was the source of beta-2 trf for our sensitivity and specificity studies. Transferrin was measured by rate nephelometry. RESULTS: The 2.5-h procedure demonstrated reproducible migration (CV <2.5%) on five lots of gels. Detection of beta-2 trf at 0.002 g/L in an unconcentrated sample was attributed to reproducible application, quality of the anti-trf antiserum, and a sensitive acid violet stain. Our beta-2 trf findings (two negative and five positive) in seven available clinical samples agreed with the reference laboratory results. In 12 months after its inception, this test was ordered 48 times vs 13 in the previous year when testing was sent out. CONCLUSION: This method provides physicians with a rapid, reliable aid in the diagnosis of suspected CSF leakage, as described in a case report.

Beta-trace protein as sensitive marker for CSF rhinorhea and CSF otorhea.

OBJECTIVE: Beta-trace protein concentrations in cerebrospinal fluid (CSF), serum and nasal secretions are investigated with a new quantitative, immunonephelometric assay. RESULTS: The mean beta-trace concentration of normal lumbar CSF (18.4 mg/l) and normal serum (0.59 mg/l), from n = 132 control patients, were 10% higher than reported earlier for smaller control groups. The reference range of beta-trace protein in nasal secretions is very low (median: 0.016 mg/l, range <0.003-0.12 mg/l, for n = 29 controls). Clinically confirmed cases of CSF rhinorhea (n = 20) showed beta-trace concentrations between 0.36 and 53.6 mg/l, with a median of 2.4 mg/l. We propose a cut-off value of 0.35 mg/l above which a CSF contamination in the secretion is plausible. A clinically confirmed CSF otorhea had a value of 1.75 mg/l. CONCLUSION: This new beta-trace protein assay offers a fast, sensitive and reliable routine method to detect a CSF rhinorhea or otorhea.

[Analysis of beta-"trace" protein detects leakage of cerebrospinal fluid. Quickly, safely and cheaply]. / Analys av beta-"trace"-protein spårar läckage av cerebrospinalvätska. Snabbt, säkert och billigt.

beta-trace protein is an immunological marker for the detection of cerebrospinal fluid leakage to the nose or ear. Analysis of beta-trace protein via a nephelometric assay is a valuable test for the identification of cerebrospinal fluid in rhinorrhea or otorrhea.

[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.