Validation of assembled nucleic acid-based tests in diagnostic microbiology laboratories

Medical microbiology and virology laboratories use nucleic acid tests (NAT) to detect genomic material of infectious organisms in clinical samples. Laboratories choose to perform assembled (or in-house) NAT if commercial assays are not available or if assembled NAT are more economical or accurate. One reason commercial assays are more expensive is because extensive validation is necessary before the kit is marketed, as manufacturers must accept liability for the performance of their assays, assuming their instructions are followed. On the other hand, it is a particular laboratory's responsibility to validate an assembled NAT prior to using it for testing and reporting results on human samples. There are few published guidelines for the validation of assembled NAT. One procedure that laboratories can use to establish a validation process for an assay is detailed in this document. Before validating a method, laboratories must optimise it and then document the protocol. All instruments must be calibrated and maintained throughout the testing process. The validation process involves a series of steps including: (i) testing of dilution series of positive samples to determine the limits of detection of the assay and their linearity over concentrations to be measured in quantitative NAT; (ii) establishing the day-to-day variation of the assay's performance; (iii) evaluating the sensitivity and specificity of the assay as far as practicable, along with the extent of cross-reactivity with other genomic material; and (iv) assuring the quality of assembled assays using quality control procedures that monitor the performance of reagent batches before introducing new lots of reagent for testing.     

An open-label trial of divalproex in children and adolescents with bipolar disorder

OBJECTIVE: This study evaluated the safety and effectiveness of divalproex sodium (Depakote ) in the treatment of youths with bipolar disorder. METHOD: Forty bipolar disorder patients aged 7 to 19 years, with a manic, hypomanic, or mixed episode, enrolled in an open-label study of divalproex (2-8 weeks), followed by a double-blind, placebo-controlled period (8 weeks). RESULTS: Twenty-two subjects (61%) showed > or =50% improvement in Mania Rating Scale (MRS) scores during the open-label period. Significant ( <.001) improvements from baseline were seen for mean scores of all efficacy measures, including the MRS, Manic Syndrome Scale, Behavior and Ideation Scale, Brief Psychiatric Rating Scale, Clinical Global Impressions Severity scale, and Hamilton Rating Scale for Depression. Of the 23 subjects who discontinued the study during the open-label period, 6 (15%) discontinued for ineffectiveness, 6 (15%) for intolerance, 6 (15%) for noncompliance, and 6 (15%) for other reasons. Adverse events were generally mild or moderate in severity, with the most common being headache, nausea, vomiting, diarrhea, and somnolence. Laboratory data results were unremarkable. Too few subjects participated in the double-blind period for statistical analysis. CONCLUSION: This study provides preliminary support for the safety and effectiveness of divalproex in the treatment of bipolar disorder in youths.     

Thrombectomy in posterior circulation stroke through persistent primitive trigeminal artery: A case report

We describe a case of intra-arterial treatment (IAT) of acute posterior circulation occlusion in a patient with a persistent primitive trigeminal artery (PPTA). The patient presented with an acute left sided hemiparesis and loss of consciousness (Glasgow coma score of 5). Computed tomography angiography showed an acute occlusion of the right internal carotid artery (ICA), the PPTA, distal basilar artery (BA), right posterior cerebral artery (PCA), and right superior cerebellar artery (SCA). Stent-retriever assisted thrombectomy was not considered possible through the hypoplastic proximal BA. After passage of the proximal ICA occlusion, the right PCA and SCA were recanalized through the PPTA, with a single thrombectomy procedure. Ten days after intervention patient was discharged scoring optimal EMV with only a mild facial and left hand paresis remaining. PPTA is a persistent embryological carotid–basilar connection. Knowledge of existing (embryonic) variants in neurovascular anatomy is essential when planning and performing acute neurointerventional procedures.     

Characterization and prediction of rate-dependent flexibility in lumbar spine biomechanics at room and body temperature

Background contextThe soft tissues of the spine exhibit sensitivity to strain-rate and temperature, yet current knowledge of spine biomechanics is derived from cadaveric testing conducted at room temperature at very slow, quasi-static rates.PurposeThe primary objective of this study was to characterize the change in segmental flexibility of cadaveric lumbar spine segments with respect to multiple loading rates within the range of physiologic motion by using specimens at body or room temperature. The secondary objective was to develop a predictive model of spine flexibility across the voluntary range of loading rates.Study designThis in vitro study examines rate- and temperature-dependent viscoelasticity of the human lumbar cadaveric spine.MethodsRepeated flexibility tests were performed on 21 lumbar function spinal units (FSUs) in flexion-extension with the use of 11 distinct voluntary loading rates at body or room temperature. Furthermore, six lumbar FSUs were loaded in axial rotation, flexion-extension, and lateral bending at both body and room temperature via a stepwise, quasi-static loading protocol. All FSUs were also loaded using a control loading test with a continuous-speed loading-rate of 1-deg/sec. The viscoelastic torque-rotation response for each spinal segment was recorded. A predictive model was developed to accurately estimate spine segment flexibility at any voluntary loading rate based on measured flexibility at a single loading rate.ResultsStepwise loading exhibited the greatest segmental range of motion (ROM) in all loading directions. As loading rate increased, segmental ROM decreased, whereas segmental stiffness and hysteresis both increased; however, the neutral zone remained constant. Continuous-speed tests showed that segmental stiffness and hysteresis are dependent variables to ROM at voluntary loading rates in flexion-extension. To predict the torque-rotation response at different loading rates, the model requires knowledge of the segmental flexibility at a single rate and specified temperature, and a scaling parameter. A Bland-Altman analysis showed high coefficients of determination for the predictive model.ConclusionsThe present work demonstrates significant changes in spine segment flexibility as a result of loading rate and testing temperature. Loading rate effects can be accounted for using the predictive model, which accurately estimated ROM, neutral zone, stiffness, and hysteresis within the range of voluntary motion.     

Characterization of recombinant human bone morphogenetic protein‐2 delivery from injectable hyaluronan‐based hydrogels by means of 125I‐radiolabelling

This study presents a thorough in vitro and in vivo characterization of the delivery of bone morphogenetic protein 2 (BMP‐2) from a hyaluronan‐based hydrogel system. The in vitro release of BMP‐2 from similar hydrogels has previously been studied by enzyme‐linked immunosorbent assay (ELISA), by which only a fraction of the loaded protein is detected. In the current study, ¹²⁵I radiolabelling was used instead to monitor BMP‐2 in vitro and in vivo. To minimize protein loss during handling, ¹²⁵I‐BMP‐2 adsorption to different tubes was studied at different times and temperatures. The data showed that Protein LoBind tubes exhibited the lowest protein affinity. Furthermore, a biphasic release profile of biologically active BMP‐2 was observed both in vitro and in vivo, with the initial fast phase during the first week, followed by a slower release during the remaining 3 weeks. The initial fast‐release phase corresponded to the early bone formation observed after 8 days in an ectopic model in rats. Bone volume and mineral content increased until day 14, after which a decrease in bone volume was observed, possibly due to resorption in response to decreased amounts of released BMP‐2. Overall, the results suggested that cautious protein handling and a reliable quantification technique are essential factors for successful design of a BMP‐2 delivery system. Copyright © 2012 John Wiley & Sons, Ltd.     

Gamma Knife radiosurgery of olfactory groove meningiomas provides a method to preserve subjective olfactory function

Anosmia is a common outcome after resection of olfactory groove meningioma(s) (OGM) and for some patients represents a significant disability. To evaluate long term tumor control rates and preservation of subjective olfaction after Gamma Knife (GK) stereotactic radiosurgery (SRS) of OGM. We performed a retrospective chart review and telephone assessments of 41 patients who underwent GK SRS between 1987 and 2008. Clinical outcomes were stratified by full, partial or no subjective olfaction, whereas tumor control was assessed by changes in volume greater or lesser than 25 %. The median clinical and imaging follow-up were 76 and 65 months, respectively. Prior to SRS, 19 (46 %) patients had surgical resections and two (5 %) had received fractionated radiation therapy. Twenty four patients (59 %) reported a normal sense of smell, 12 (29 %) reported a reduced sense of smell and five (12 %) had complete anosmia. The median tumor volume was 8.5 cm³ (range 0.6–56.1), the mean radiation dose at the tumor margin was 13 Gy (range 10–20) and the median estimated dose to the olfactory nerve was 5.1 Gy (range 1.1–18.1). At follow-up, 27 patients (66 %) reported intact olfaction (three (7 %) described return to a normal sense of smell), nine (22 %) described partial anosmia, and five (12 %) had complete anosmia. No patient reported deterioration in olfaction after SRS. Thirteen patients (32 %) showed significant tumor regression, 26 (63 %) had no further growth and two (5 %) had progressed. The progression free tumor control rates were 97 % at 1 year and 95 % at 2, 10 and 20 years. Symptomatic adverse radiation effects occurred in three (7 %) patients. Stereotactic radiosurgery provided both long term tumor control and preservation of olfaction.