Citations

Abstract Background Scientists working in the field of xenotransplantation do not employ a uniform method to measure and report natural and induced antibody responses to non‐ G alα(1,3) G al (non‐ G al) epitopes. Such humoral responses are thought to be particularly pathogenic after transplantation of vascularized Gal TKO pig organs and having a more uniform assay and reporting format would greatly facilitate comparisons between laboratories. Methods Flow cytometry allows examination of antibody reactivity to intact antigens in their natural location and conformation on cell membranes. We have established a simple and reproducible flow cytometric assay to detect antibodies specific for non‐ G al pig antigens using primary porcine aortic endothelial cells ( pAEC s) and cell culture‐adapted pAEC cell lines generated from wild type and α1,3galactosyl transferase knockout (Gal TKO ) swine. Results The consensus protocol we propose here is based on procedures routinely used in four xenotransplantation centers and was independently evaluated at three sites using shared cells and serum samples. Our observation support use of the cell culture‐adapted Gal TKO pAEC KO :15502 cells as a routine method to determine the reactivity of anti‐non‐ G al antibodies in human and baboon serum. Conclusions We have developed an assay that allows the detection of natural and induced non‐Gal xenoreactive antibodies present in human or baboon serum in a reliable and consistent manner. This consensus assay and format for reporting the data should be accessible to laboratories and will be useful for assessing experimental results between multiple research centers. Adopting this assay and format for reporting the data should facilitate the detection, monitoring, and detailed characterization of non‐ G al antibody responses.

Abstract Background Xenograft rejection of pigs organs with an engineered mutation in the GGTA‐1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non‐Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNA s which encode pig antigens that bind to IgG induced after pig‐to‐primate cardiac xenotransplantation. One of these gene products was a glycosyltransferase with homology to the bovine β1,4 N‐acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance. Methods The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced, and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK‐B4T) was produced. Expression of porcine B4GALNT2 in HEK‐B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK‐B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK‐B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non‐Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining. Results The porcine B4 GALNT 2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4 GALNT 2 genes, respectively. The B4 GALNT 2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4 GALNT 2 in human HEK cells ( HEK ‐B4T) results in increased binding of antibody to the B4 GALNT 2 enzyme, and increased reactivity with anti‐Sd a and DBA . HEK ‐B4T cells show increased sensitivity to complement mediated lysis when challenged with serum from primates after pig to primate cardiac xenotransplantation. In GTKO and GTKO : CD 55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non‐Gal antibody, measured using GTKO PAEC s, and the induction of antibodies which preferentially bind to HEK ‐B4T cells. Conclusion The functional isolation of the porcine B4 GALNT 2 gene from a PAEC expression library, the pattern of B4 GALNT 2 gene expression and its sensitization of HEK ‐B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4 GALNT 2 produces a new immunogenic non‐Gal glycan which contributes in part to the non‐Gal immune response detected after pig‐to‐baboon cardiac xenotransplantation.

Intramedullary ependymomas are surgically curable tumors. However, their surgical resection poses several challenges. In this intraoperative video we illustrate the main steps for the surgical resection of a cervical intramedullary ependymoma. These critical steps include: adequate exposure of the entire length of the tumor; use of the intraoperative ultrasound; identification of the posterior median sulcus and separation of the posterior columns; Identification of the plane between the spinal cord and the tumor; mobilization and debulking of the tumor and disconnection of the vascular supply (usually from small anterior spinal artery branches). Following these basic steps a complete resection can be safely achieved in many cases. The video can be found here: http://youtu.be/QMYXC_F4O4U .

A long-held dogma in neurosurgery is that parenchymal arteriovenous malformations (AVMs) are congenital. However, there is no strong evidence supporting this theory. An increasing number of documented cases of de novo formation of parenchymal AVMs cast doubt on their congenital nature and suggest that indeed the majority of these lesions may form after birth. Further evidence suggesting the postnatal development of parenchymal AVMs comes from the exceedingly rare diagnosis of these lesions in utero despite the widespread availability of high-resolution imaging modalities such as ultrasound and fetal MRI. The exact mechanism of AVM formation has yet to be elucidated, but most likely involves genetic susceptibility and environmental triggering factors. In this review, the authors report 2 cases of de novo AVM formation and analyze the evidence suggesting that they represent an acquired condition.

Context.— The Q-Tracks program, created in 1999, is a quality monitoring subscription service offered by the College of American Pathologists. Objective.— To establish benchmarks in quality metrics, monitor changes in performance over time, and identify practice characteristics associated with better performance. Design.— The Q-Tracks program provides ongoing study of multiple metrics offered in most laboratory disciplines. The design enables measuring the effects of process changes and comparisons with other participating laboratories. Each laboratory Q-Tracks monitor has a primary quality indicator and additional secondary indicators. Results.— To date, 19 Q-Tracks monitors have been offered, with 12 currently active monitors. Q-Tracks are primarily conducted in hospital-based laboratories in the United States, Canada, and 21 other countries. Common to most Q-Tracks monitors is a demonstration of performance improvement by subscribers with long-term participation. This finding was seen in preanalytic, turnaround time, and postanalytic measures. Q-Tracks monitors contribute to the overall demonstration and improvement of laboratory and hospital quality because they address core quality measures for the College of American Pathologists Laboratory Accreditation Program and multiple Joint Commission National Patient Safety Goals. Conclusions.— The Q-Tracks program has established multiple benchmarks in most disciplines of the laboratory and has demonstrated significant performance improvement in benchmarks and individual laboratories over time.

Context.— Both the regulations in the Clinical Laboratory Improvement Amendments of 1988 (CLIA) and the checklists of the College of American Pathologists (CAP) Laboratory Accreditation Program require clinical laboratories to verify performance characteristics of quantitative test systems. Laboratories must verify performance claims when introducing an unmodified, US Food and Drug Administration–cleared or approved test system, and they must comply with requirements for periodic calibration and calibration verification for existing test systems. They must also periodically verify the analytical measurement range of many quantitative test systems. Objective.— To provide definitions for many of the terms used in these regulations, to describe a set of basic analyses that laboratories may adapt to demonstrate compliance with both CLIA and the CAP Laboratory Accreditation Program checklists for performing calibration verification and for verifying the analytical measurement range of test systems, to review some of the recommended procedures for establishing performance goals, and to provide data illustrating the performance goals used in some of the CAP's calibration verification and linearity surveys. Data Sources.— The CAP's calibration verification and linearity survey programs, the CLIA regulations, the Laboratory Accreditation Program requirements, and published literature were used to meet these objectives. Conclusions.— Calibration verification and linearity and analytical measurement range verification should be performed using suitable materials with assessment of results using well-defined evaluation protocols. We describe the CAP's calibration verification and linearity programs that may be used for these purposes.

Object The aim of this study was to determine age-related differences in short-term (1-year) outcomes in patients with unruptured intracranial aneurysms (UIAs). Methods Four thousand fifty-nine patients prospectively enrolled in the International Study of Unruptured Intracranial Aneurysms were categorized into 3 groups by age at enrollment: < 50, 50–65, and > 65 years old. Outcomes assessed at 1 year included aneurysm rupture rates, combined morbidity and mortality from aneurysm procedure or hemorrhage, and all-cause mortality. Periprocedural morbidity, in-hospital morbidity, and poor neurological outcome on discharge (Rankin scale score of 3 or greater) were assessed in surgically and endovascularly treated groups. Univariate and multivariate associations of each outcome with age were tested. Results The risk of aneurysmal hemorrhage did not increase significantly with age. Procedural and in-hospital morbidity and mortality increased with age in patients treated with surgery, but remained relatively constant with increasing age with endovascular treatment. Poor neurological outcome from aneurysm- or procedure-related morbidity and mortality did not differ between management groups for patients 65 years old and younger, but was significantly higher in the surgical group for patients older than 65 years: 19.0% (95% confidence interval [CI] 13.9%–24.4%), compared with 8.0% (95% CI 2.3%–13.6%) in the endovascular group and 4.2% (95% CI 2.3%–6.2%) in the observation group. All-cause mortality increased steadily with increasing age, but differed between treatment groups only in patients < 50 years of age, with the surgical group showing a survival advantage at 1 year. Conclusions Surgical treatment of UIAs appears to be safe, prevents 1-year hemorrhage, and may confer a survival benefit in patients < 50 years of age. However, surgery poses a significant risk of morbidity and death in patients > 65 years of age. Risk of endovascular treatment does not appear to increase with age. Risks and benefits of treatment in older patients should be carefully considered, and if treatment is deemed necessary for patients older than 65 years, endovascular treatment may be the best option.