An improved size exclusion-HPLC method for molecular size distribution analysis of immunoglobulin G using sodium perchlorate in the eluent.

Size exclusion (SE) high performance liquid chromatography (HPLC) is widely used for the molecular size distribution (MSD) analyses of various therapeutic proteins. We report development and validation of a SE-HPLC method for MSD analyses of immunoglobulin G (IgG) in products using a TSKgel SuperSW3000 column and eluting it with 0.4M NaClO4, a chaotropic salt, in 40mM phosphate buffer, pH 6.8. The chromatograms show distinct peaks of aggregates, tetramer, and two dimers, as well as the monomer and fragment peaks. In addition, the method offers about half the run time (12min), better peak resolution, improved peak shape and more stable base-line compared to HPLC methods reported in the literature, including that in the European Pharmacopeia (EP). A comparison of MSD analysis results between our method and the EP method shows interactions between the protein and the stationary phase and partial adsorption of aggregates and tetramer on the stationary phase, when the latter method is used. Thus, the EP method shows lower percent of aggregates and tetramer than are actually present in the products. In view of the fact that aggregates have been attributed to playing a critical role in adverse reactions due to IgG products, our observation raises a major concern regarding the actual aggregate content in these products since the EP method is widely used for MSD analyses of IgG products. Our method eliminates (or substantially reduces) the interactions between the proteins and stationary phase as well as the adsorption of proteins onto the column. Our results also show that NaClO4 in the eluent is more effective in overcoming the protein/column interactions compared to Arg-HCl, another chaotropic salt. NaClO4 is shown not to affect the molecular size and relative distribution of different molecular forms of IgG. The method validated as per ICH Q2(R1) guideline using IgG products, shows good specificity, accuracy, precision and a linear concentration dependence of peak areas for different molecular forms. In summary, our method gives more reliable results than the SE-HPLC methods for MSD analyses of IgG reported in the literature, including the EP, particularly for aggregates and tetramer. The results are interpreted in terms of ionic (polar) and hydrophobic interactions between the stationary phase and the IgG protein.

Protein Nitrogen Determination by Kjeldahl Digestion and Ion Chromatography.

We report development and validation of a simple, rapid, and accurate method for the quantitation of protein nitrogen, which combines Kjeldahl digestion and ion chromatography with suppressed conductivity detection and requires nanomolar amount of nitrogen in samples (≥10 µg protein). The mechanism of suppressed conductivity detection does not permit analysis of samples containing copper (present in Kjeldahl digestion solution) and aluminum (present in many vaccines as adjuvants) due to precipitation of their hydroxides within the suppressor. We overcame this problem by including 10 µM oxalic acid in Kjeldahl digests and in the eluent (30 mM methanesulfonic acid). The chromatography is performed using an IonPac CS-16 cation exchange column by isocratic elution. The method reduces the digestion time to less than 1 h and eliminates the distillation and titration steps of the Kjeldahl method, thereby reducing the analysis time significantly and improving precision and accuracy. To determine protein nitrogen in samples containing non-protein nitrogen, proteins are precipitated by a mixture of deoxycholate and trichloroacetic acid and the precipitates are analyzed after dissolving in KOH. The method is particularly useful for biological samples that are limited and can also be applied to food, environmental, and other materials.

A novel approach for preparation of the antisera reagent for potency determination of inactivated H7N9 influenza vaccines.

BACKGROUND: The potency of inactivated influenza vaccines is determined using a single-radial immunodiffusion (SRID) assay and requires standardized reagents consisting of a Reference Antigen and an influenza strain-specific antiserum. Timely availability of reagents is a critical step in influenza vaccine production, and the need for backup approaches for reagent preparation is an important component of pandemic preparedness. OBJECTIVES: When novel H7N9 viruses emerged in China in 2013, candidate inactivated H7N9 influenza vaccines were developed for evaluation in clinical trials, and reagents were needed to measure vaccine potency. METHODS: We previously described an alternative approach for generating strain-specific potency antisera, utilizing modified vaccinia virus Ankara vectors to produce influenza hemagglutinin (HA)-containing virus-like particles (VLPs) for immunization. Vector-produced HA antigen is not dependent upon the success of the traditional bromelain-digestion and HA purification. RESULTS: Antiserum for H7N9 vaccines, produced after immunization of sheep with preparations of bromelain-HA (br-HA), was not optimal for the SRID assay, and the supply of antiserum was limited. However, antiserum obtained from sheep boosted with VLPs containing H7 HA greatly improved the ring quality in the SRID assay. Importantly, this antiserum worked well with both egg- and cell-derived antigen and was distributed to vaccine manufacturers. CONCLUSIONS: Utilizing a previously developed approach for preparing vaccine potency antiserum, we have addressed a major bottleneck encountered in preparation of H7N9 vaccine reagents. The combination of br-HA and mammalian VLPs for sequential immunization represents the first use of an alternative approach for producing an influenza vaccine potency antiserum.

A cadaveric analysis of cervical fixation: the effect of intermediate fixation points and dynamization in multilevel cervical fusions.

OBJECT: The authors conducted a study to compare biomechanical effects on the cervical spine of bridging fixation and intermediate fixation techniques, in both fixed and dynamic modes. METHODS: A biaxial, servohydraulic machine biomechanically tested 23 human cervical spines for stiffness and strain in compression, extension, flexion, and lateral bending through 3 specimen states: 1) intact, 2) defect (corpectomy and discectomy), and 3) grafting with plate application in 1 of 4 constructs: C3-7 dynamized long strut (DLS), C3-7 fixed long strut (FLS), C3-5-7 dynamized multisegment (DMS), and C3-5-7 fixed multisegment (FMS). RESULTS: Compared with FMS, FLS had significantly greater strain in extension (at C-3 and at the rostral and caudal parts of the graft) and in lateral bending (at C-3 and at the caudal part of the graft). Fixed (FLS and FMS) constructs had greater flexion stiffness than did dynamized (DLS and DMS) constructs and showed a trend toward greater lateral bending stiffness. Instrumentation revealed greater extension strain with the long fixed (FLS and DLS) constructs than with the multifixed (FMS and FMS) constructs at the rostral and caudal parts of the graft but no significant differences between the dynamized (DLS and DMS) and fixed (FLS and FMS) constructs. CONCLUSIONS: Multisegmental fixation provided greater stabilizing forces than did bridging constructs for both dynamized and fixed plates. Use of multisegmental fixation can potentially decrease strain at the screw-plate interface and reduce the rate of hardware failure.

Role of patient information handouts following operative treatment of ankle fractures: a prospective randomized study.

BACKGROUND: Widespread evidence exists for directed patient information interventions (eg, pamphlets) in the setting of several orthopaedic conditions and interventions. Up until now, no study had assessed the role of these interventions in the management of patients following ankle fractures. METHODS: Between 2005 and 2007, 40 patients who suffered an operative ankle fracture were randomized to either a standard treatment group for an ankle fracture or an enhanced information group who received an American Academy of Orthopaedic Surgeons ankle fracture information pamphlet that explained postoperative routine at our institution and a physiotherapy handout depicting a standard protocol. Study participants were followed for 3 months clinically and radiographically. At the 6-week and 3-month intervals, study participants completed the Olerud-Molander Questionnaire and 2 questions regarding their level of satisfaction. The primary outcome measure was the Likert-scale-based survey question determining the level of satisfaction with the treating staff. RESULTS: Participants in the enhanced information group were more satisfied with treatment at 3 months (9.2 vs 6.3; P < .001). There were significant improvements in work/activity ability at 6 weeks (P = .01), but this advantage disappeared at 3 months (P = .24). No differences in postoperative complication rates were noted. CONCLUSIONS: Information enhancement in the form of pamphlets can be helpful in providing patients with accessible information in the postoperative period. While they do not seem to have a sustained impact on postoperative outcomes, handouts may enhance the interaction between staff and patient at postoperative visits, improving patient satisfaction. LEVEL OF EVIDENCE: Level I, appropriately powered randomized prospective cohort study.

Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products.

Most biological products, including vaccines, administered by the parenteral route are required to be tested for sterility at the final container and also at various stages during manufacture. The sterility testing method described in the Code of Federal Regulations (21 CFR 610.12) and the United States Pharmacopoeia (USP, Chapter <71>) is based on the observation of turbidity in liquid culture media due to growth of potential contaminants. We evaluated rapid microbiological methods (RMM) based on detection of growth 1) by adenosine triphosphate (ATP) bioluminescence technology (Rapid Milliflex(®) Detection System [RMDS]), and 2) by CO(2) monitoring technologies (BacT/Alert and the BACTEC systems), as alternate sterility methods. Microorganisms representing Gram negative, Gram positive, aerobic, anaerobic, spore forming, slow growing bacteria, yeast, and fungi were prepared in aliquots of Fluid A or a biological matrix (including inactivated influenza vaccines) to contain approximately 0.1, 1, 10 and 100 colony forming units (CFU) in an inoculum of 10 ml. These preparations were inoculated to the specific media required for the various methods: 1) fluid thioglycollate medium (FTM) and tryptic soy broth (TSB) of the compendial sterility method (both membrane filtration and direct inoculation); 2) tryptic soy agar (TSA), Sabouraud dextrose agar (SDA) and Schaedler blood agar (SBA) of the RMDS; 3) iAST and iNST media of the BacT/Alert system and 4) Standard 10 Aerobic/F and Standard Anaerobic/F media of the BACTEC system. RMDS was significantly more sensitive in detecting various microorganisms at 0.1CFU than the compendial methods (p<0.05), whereas the compendial membrane filtration method was significantly more sensitive than the BACTEC and BacT/Alert methods (p<0.05). RMDS detected all microorganisms significantly faster than the compendial method (p<0.05). BacT/Alert and BACTEC methods detected most microorganisms significantly faster than the compendial method (p<0.05), but took almost the same time to detect the slow growing microorganism P. acnes, compared to the compendial method. RMDS using SBA detected all test microorganisms in the presence of a matrix containing preservative 0.01% thimerosal, whereas the BacT/Alert and BACTEC systems did not consistently detect all the test microorganisms in the presence of 0.01% thimerosal. RMDS was compatible with inactivated influenza vaccines and aluminum phosphate or aluminum hydroxide adjuvants at up to 8 mg/ml without any interference in bioluminescence. RMDS was shown to be acceptable as an alternate sterility method taking 5 days as compared to the 14 days required of the compendial method. Isolation of microorganisms from the RMDS was accomplished by re-incubation of membranes with fresh SBA medium and microbial identification was confirmed using the MicroSEQ Identification System. BacT/Alert and BACTEC systems may be applicable as alternate methods to the compendial direct inoculation sterility method for products that do not contain preservatives or anti-microbial agents.

Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines.

Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009-2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified.

A protein multiplex microarray substrate with high sensitivity and specificity.

The problems that have been associated with protein multiplex microarray immunoassay substrates and existing technology platforms include: binding, sensitivity, a low signal to noise ratio, target immobilization and the optimal simultaneous detection of diverse protein targets. Current commercial substrates for planar multiplex microarrays rely on protein attachment chemistries that range from covalent attachment to affinity ligand capture, to simple adsorption. In this pilot study, experimental performance parameters for direct monoclonal mouse IgG detection were compared for available two and three-dimensional slide surface coatings with a new colloidal nitrocellulose substrate. New technology multiplex microarrays were also developed and evaluated for the detection of pathogen-specific antibodies in human serum and the direct detection of enteric viral antigens. Data supports the nitrocellulose colloid as an effective reagent with the capacity to immobilize sufficient diverse protein target quantities for increased specific signal without compromising authentic protein structure. The nitrocellulose colloid reagent is compatible with the array spotters and scanners routinely used for microarray preparation and processing. More importantly, as an alternate to fluorescence, colorimetric chemistries may be used for specific and sensitive protein target detection. The advantages of the nitrocellulose colloid platform indicate that this technology may be a valuable tool for the further development and expansion of multiplex microarray immunoassays in both the clinical and research laboratory environment.

Maximizing outpatient computed tomography productivity using multiple technologists.

PURPOSE: A key radiology stakeholder demand is to increase patient access to computed tomography (CT) and reduce waiting lists. However, the number of patients that a single technologist can scan is limited because of the many tasks required to process a patient through a CT scan. However, many tasks could be performed simultaneously by using additional personnel. This study evaluated how many additional patients can be scanned using a 2- or 3-technologist model with outpatient multidetector CT and its impact on CT capacity. METHODS: The number and type of individual technologist tasks were initially evaluated. The time to perform these tasks was then measured using 1-, 2-, and 3-technologist models, including the time a patient was within the CT scanner room, to determine the hourly patient throughput on a CT scanner. Two theoretic CT operations were then developed to evaluate the impact on CT capacity. RESULTS: Thirty-four technologist workflow tasks were identified. A total of 205 outpatients were evaluated. The total time to perform all tasks for 1-, 2-, and 3-technologist models was 27, 23, and 22 minutes, respectively. CT room time per patient for 1-, 2-, and 3-technologist models was 12, 9.7, and 8.0 minutes, respectively. However, the number of patients scanned per hour for 1-, 2-, and 3-technologist models was 2.2, 5.2, and 7.5, respectively. There was an increase of more than 12,000 potential patient CT slots made available using 2 technologists 7 days per week and 22,000 additional slots for a 3-technologist model when compared with a single-technologist model on weekdays only. CONCLUSION: A single-technologist model for outpatient multidetector CT is inefficient with limited opportunity for increased patient throughput. The use of multiple technologists (or other key personnel) optimizes CT throughput and capacity, particularly with a 3-technologist model, which can yield a greater than three-fold increase in CT productivity.

Topical triple-antibiotic ointment as a novel therapeutic choice in wound management and infection prevention: a practical perspective.

Triple-antibiotic ointment (TAO) is a safe and effective topical agent for preventing infections in minor skin trauma. The formulation contains neomycin, polymyxin B and bacitracin in a petrolatum base. TAO is active against the most common disease-causing pathogens found in wounds and on the skin and may be an attractive alternative to oral therapy in select circumstances. Resistance to TAO does not develop readily, and safety studies have shown that the risk of allergic sensitivity to TAO is low. Susceptibility profiles of TAO have remained relatively unchanged since its discovery. Prophylaxis or treatment with TAO should be considered as resistant organisms continue to emerge in the community and hospital setting.

Epsin N-terminal homology domains perform an essential function regulating Cdc42 through binding Cdc42 GTPase-activating proteins.

Epsins are endocytic proteins with a structured epsin N-terminal homology (ENTH) domain that binds phosphoinositides and a poorly structured C-terminal region that interacts with ubiquitin and endocytic machinery, including clathrin and endocytic scaffolding proteins. Yeast has two redundant genes encoding epsins, ENT1 and ENT2; deleting both genes is lethal. We demonstrate that the ENTH domain is both necessary and sufficient for viability of ent1Deltaent2Delta cells. Mutational analysis of the ENTH domain revealed a surface patch that is essential for viability and that binds guanine nucleotide triphosphatase-activating proteins for Cdc42, a critical regulator of cell polarity in all eukaryotes. Furthermore, the epsins contribute to regulation of specific Cdc42 signaling pathways in yeast cells. These data support a model in which the epsins function as spatial and temporal coordinators of endocytosis and cell polarity.

The maximally exposed individual--comparison of maximum likelihood estimation of high quantiles to an extreme value estimate.

The dose to human and nonhuman individuals inflicted by anthropogenic radiation is an important issue in international and domestic policy. The current paradigm for nonhuman populations asserts that if the dose to the maximally exposed individuals in a population is below a certain criterion (e.g., <10 mGy d(-1)) then the population is adequately protected. Currently, there is no consensus in the regulatory community as to the best statistical approach. Statistics, currently considered, include the maximum likelihood estimator for the 95th percentile of the sample mean and the sample maximum. Recently, the investigators have proposed the use of the maximum likelihood estimate of a very high quantile as an estimate of dose to the maximally exposed individual. In this study, we compare all of the above-mentioned statistics to an estimate based on extreme value theory. To determine and compare the bias and variance of these statistics, we use Monte Carlo simulation techniques, in a procedure similar to a parametric bootstrap. Our results show that a statistic based on extreme value theory has the least bias of those considered here, but requires reliable estimates of the population size. We recommend establishing the criterion based on what would be considered acceptable if only a small percentage of the population exceeded the limit, and hence recommend using the maximum likelihood estimator of a high quantile in the case that reliable estimates of the population size are not available.

Coarsening of fractal viscous fingering patterns.

We have measured the coarsening due to surface tension of radially grown fractal viscous fingering patterns. The patterns at late times depend on the structural form at the onset of coarsening, providing information on the age of the fractal. The coarsening process is not dynamically scale invariant, exhibiting two dynamic length scales that grow as L1(t) approximately t(0.22+/-0.02) and L2(t) approximately t(0.31+/-0.02). The measured exponents are in agreement with the results of recent numerical studies of diffusion-controlled coarsening of a diffusion-limited aggregation fractal [Phys. Rev. E 65, 050501 (2002)]].

Risk factors and prevalence of injuries in horses during various types of steeplechase races.

OBJECTIVE: To identify the types of injuries sustained by horses that competed in steeplechase races and determine the prevalence of and risk factors for those injuries. DESIGN: Prospective study. ANIMALS: 2,680 horses that competed in various types of steeplechase races from 1996 through 2000. PROCEDURE: Data regarding races; environment; equipment problems; the number of horses that entered, started, and finished races; the number of riders that fell; and the number of horses that were slowed or stopped by the rider, ran off the course, fell, and sustained injuries or physical abnormalities during races were collected on a standard form by the official veterinarian who attended each meet. Data from all meets were not recorded; however, in recorded meets, data from every race were reported. RESULTS: Data for 197 hurdle, 65 timber, 76 flat, and 8 mixed races were recorded. Nine (3.4/1,000 horses that started in races) horses died or were euthanatized, and 7 of those were associated with catastrophic musculoskeletal injury. Seven fractures were recorded. Four fractures involved forelimbs, 1 involved a hind limb, and 2 involved the cervical portion of the vertebral column. All horses with fractures were euthanatized. Deep or hard course conditions were associated with an increased risk of breakdown injuries. CONCLUSIONS AND CLINICAL RELEVANCE: Successful development and implementation of strategies to prevent injuries and death in horses in steeplechase races depend on a clear understanding of the types and prevalence of injuries involved and risk factors associated with those injuries.

Cervical spondylotic myelopathy: make the difficult diagnosis, then refer for surgery.

Cervical spondylotic myelopathy is the result of narrowing of the cervical spinal canal by degenerative and congenital changes. Prompt surgical treatment is key, but the diagnosis can be difficult because the signs and symptoms can vary widely and there are no pathognomonic findings.

Pseudobulbar palsy caused by a large petroclival meningioma: report of two cases.

Two patients sought treatment for symptoms of bulbar motor dysfunction and the marked emotional lability that characterizes pseudobulbar palsy (PBP). Magnetic resonance (MR) imaging showed large petroclival masses with severe compression and displacement of the brainstem. A suboccipital craniectomy with a transpetrosal, transtentorial approach to the tumor was performed in both patients. One patient required a second-stage surgery to resect an extension of the tumor into the cavernous sinus. In both patients pathological evaluation confirmed the diagnosis of syncytial meningioma. After surgery, PBP resolved in both patients. Large posterior fossa tumors should be included in the differential diagnosis of patients with a clinical picture of bulbar motor dysfunction coupled with emotional lability.

Standardized health check data from community-dwelling elderly people: the potential for comparing populations and estimating need.

The main aim of this study was to compare EASY-Care data obtained during nurse-administered annual health checks in two populations of older people. A secondary aim was to determine whether a standardized assessment system administered as part of routine practice by a trained nurse during the over-75 health check could generate useful information for comparing population health and functional status of community-dwelling-older people. One hundred and seventy-nine elderly people (aged 75 years and over) from the Woodstock ward, Belfast, having relatively high deprivation; and 238 elderly people from south Hampshire, ranging from affluent wards in New Forest to inner city wards, were assessed using the EASY-Care assessment system as part of their annual health check. There was a high response rate to the standardized assessment in both populations (75% and 79%). Compared to people in south Hampshire, the people in Belfast had higher relative risk of having fair/poor self-rated health, and lower relative risk of having good/sufficient accommodation and of having difficulty chewing. People in Belfast had a higher relative risk of being dependent for six of the seven IADL items and for continence of urine, bathing, grooming, use of the stairs and dressing among the ADL items. The results demonstrate the ability of data generated by assessment system to discriminate between populations of older people when used as part of routine practice. Differences in health and functional status may be associated with deprivation. Data collected during the annual health check about the health and functional status of older people could provide a useful adjunct to census and survey data to measure population needs and to support locality planning.