Determination of cerebrospinal fluid leakage by selective deletion of transferrin glycoform using an immunochromatographic assay
Cerebrospinal fluid (CSF) leakage can lead to brain and spine pathologies and there is an urgent need for a rapid diagnostic method for determining CSF leakage. Beta-2 transferrin (Î²2TF), asialotransferrin, is a specific CSF glycoprotein biomarker used to determine CSF leakage when distinguished from serum sialotransferrin (sTF). Methods: We detected Î²2TF using an immunochromatographic assay (ICA), which can be potentially developed as a point-of-care (POC) testing platform. Sialic acid-specific lectin selectively captures sTF in multiple deletion lines within an ICA test strip, enabling the detection of Î²2TF. A sample pre-treatment process efficiently captures excess sTF increasing sensitivity for CSF leakage detection. Results: An optimal cut-off value for determining the presence of CSF in test samples was obtained from receiver operating characteristic (ROC) analysis of the ratio of the test signal intensity and the deletion lines. On 47 clinical samples, ICA test strips discriminated CSF positive from negative samples with statistically significant (positive versus negative t-test; P =0.00027). Additional artificial positive samples, prepared by mixing CSF positive and negative clinical samples, were used as a further challenge. These positive samples were clearly discriminated from the negative samples (mixture versus negative t-test; P =0.00103) and CSF leakage was determined with 97.1% specificity and 96.2% sensitivity. Conclusions: ICA represents a promising approach for POC diagnosis of CSF leakage. While requiring 70 min assay time inconvenient for POC testing, our method was significantly shorter than conventional electrophoresis-based detection methods for Î²2TF.
Detection of cerebrospinal fluid leakage by specific measurement of transferrin glycoforms
A simple and rapid detection of cerebrospinal fluid (CSF) leakage would benefit spine surgeons making critical postoperative decisions on patient care. We have assessed novel approaches to selectively determine CSF Î²2-transferrin (Î²2TF), an asialo-transferrin (aTF) biomarker, without interference from serum sialo-transferrin (sTF) in test samples. First, we performed mild periodate oxidation to selectively generate aldehyde groups in sTF for capture with magnetic hydrazide microparticles, and selective removal with a magnetic separator. Using this protocol sTF was selectively removed from mixtures of CSF and serum containing CSF aTF (Î²2TF) and serum sTF, respectively. Second, a two-step enzymatic method was developed with neuraminidase and galactose oxidase for generating aldehyde groups in sTF present in CSF and serum mixtures for magnetic hydrazide microparticle capture. After selectively removing sTF from mixtures of CSF and serum, ELISA could detect significant TF signal only in CSF, while the TF signal in serum was negligible. The new approach for selective removal of only sTF in test samples will be promising for the required intervention by a spine surgeon.
Suitability of bioresorbable cages for anterior cervical fusion
OBJECT/OBJECTIVE:The authors conducted a pilot study to determine whether a bioresorbable intervertebral fusion device composed of 85/15 polylactide-polyglycolide (PLA-PGA) copolymer packed with bone autograft is a suitable alternative to promote arthrodesis after anterior cervical discectomy (ACD) in a caprine model. METHODS:The caprine cervical spine model has been used to evaluate interbody healing and fusion after application of bone grafts and instrumentation. Whether a bioresorbable device is suitable for facilitating intervertebral bone union has not been determined. Twelve goats underwent two-level ACD and fusion; eight received bioresorbable cages packed with autologous bone, and four received autologous bone alone. Goats were maintained without an orthosis and after 12 weeks underwent physical, radiographic, and histological evaluation. Cages had structurally degraded, and two had become extruded. Stable intervertebral union developed in three (19%) of 16 cage-implanted interspaces, and one (14%) of seven bone autograft-implanted interspaces; each was judged manually to be rigid (Grade 2), radiographically to be bridged by new osseous densities (Grade 2), and histologically to have marked new bone formation (Grade 3). A primarily fibrous union, however, stabilized the cage-implanted interspaces, and eight (50%) had developed a 4 to 6-mm foreign body granuloma. These interposed soft tissues were not present in the stable autologous bone-implanted interspace, which had successfully become fused. CONCLUSIONS:Interbody cages composed of 85/15 PLA-PGA copolymer contributed to a stable fibrous union, degraded. and produced granuloma after 12 weeks. Additional evaluations are necessary to determine whether other copolymer mixtures, or other bioresorbable materials, can contribute to an arthodesis without deleterious consequences.
Enhanced vascularization and survival of neural transplants with ex vivo angiogenic gene transfer
Restoration of brain function by neural transplants is largely dependent upon the survival of donor neurons. Unfortunately, in both rodent models and human patients with Parkinson's disease the survival rate of transplanted neurons has been poor. We have employed a strategy to increase the availability of nutrients to the transplant by increasing the rate at which blood vessels are formed. Replication-deficient HSV-1 vectors containing the cDNA for human vascular endothelial growth factor (HSVhvegf) and the bacterial beta-galactosidase gene (HSVlac) have been transduced in parallel into nonadherent neuronal aggregate cultures made of cells from embryonic day 15 rat mesencephalon. Gene expression from HSVlac was confirmed in fixed preparations by staining with X-gal. VEGF expression as determined by sandwich ELISA assay of culture supernatant was up to 322-fold higher in HSVhvegf-infected than HSVlac-infected sister cultures. This peptide was also biologically active, inducing endothelial cell proliferation in vitro. Adult Sprague-Dawley rats received bilateral transplants into the striatum, with HSVlac on one side and HSVhvegf on the other. At defined intervals up to 8 weeks, animals were sacrificed and vibratome sections of the striatum were assessed for various parameters of cell survival and vascularization. Results demonstrate dose-dependent increases in blood vessel density within transplants transduced with HSVhvegf. These transplants were vascularized at a faster rate up to 4 weeks after transplantation. After 8 weeks, the average size of the HSVhvegf-infected transplants was twice that of controls. In particular, the survival of transplanted dopaminergic neurons increased 3.9-fold. Taken together these experiments provide convincing evidence that the rate of vascularization may be a major determinant of neuronal survival that can be manipulated by VEGF gene transduction.