Designing a strategy to implement cost-effective blood transfusion management in elective hip and knee arthroplasties: A study protocol

ABSTRACT: BACKGROUND: Total hip and knee arthroplasties are two of the most commonly performed procedures in orthopedic surgery. Different blood-saving measures (BSMs) are used to reduce the often-needed allogenic blood transfusions in these procedures. A recent large randomized controlled trial showed it is not cost effective to use the BSMs of erythropoietin and perioperative autologous blood salvage in elective primary hip and knee arthroplasties. Despite dissemination of these study results, medical professionals keep using these BSMs. To actually change practice, an implementation strategy is needed that is based on a good understanding of target groups and settings and the psychological constructs that predict behavior of medical professionals. However, detailed insight into these issuses is lacking. Therefore, this study aims to explore which groups of professionals should be targeted at which settings, as well as relevant barriers and facilitators that should be taken into acount in the strategy to implement evidence-based, cost-effective blood transfusion management and to de-implement BSMs. METHODS: The study consists of three phases. First, a questionnaire survey among all Dutch orthopedic hospital departments and independent treatment centers (n?=?99) will be conducted to analyze current blood management practice. Second, semistructured interviews will be held among 10 orthopedic surgeons and 10 anesthesiologists to identify barriers and facilitators that are relevant for the uptake of cost-effective blood transfusion management. Interview questions will be based on the Theoretical Domains Interview framework. The interviews will be followed by a questionnaire survey among 800 medical professionals in orthopedics and anesthesiology (400 professionals per discipline) in which the identified barriers and facilitators will be ranked by frequency and importance. Finally, an implementation strategy will be developed based on the results from the previous phases, using principles of intervention mapping and an expert panel. DISCUSSION: The developed strategy for cost-effective blood transfusion management by de-implementing BSMs is likely to reduce costs for elective hip and knee arthroplasties. In addition, this study will lead to generalized knowledge regarding relevant factors for the de-implementation of non-cost-effective interventions and insight in the differences between implementation and de-implementation strategies.     

Altered Formalin-Induced Pain and Fos Induction in the Periaqueductal Grey of Preadolescent Rats following Neonatal LPS Exposure

Animal and human studies have demonstrated that early pain experiences can produce alterations in the nociceptive systems later in life including increased sensitivity to mechanical, thermal, and chemical stimuli. However, less is known about the impact of neonatal immune challenge on future responses to noxious stimuli and the reactivity of neural substrates involved in analgesia. Here we demonstrate that rats exposed to Lipopolysaccharide (LPS; 0.05 mg/kg IP, Salmonella enteritidis) during postnatal day (PND) 3 and 5 displayed enhanced formalin-induced flinching but not licking following formalin injection at PND 22. This LPS-induced hyperalgesia was accompanied by distinct recruitment of supra-spinal regions involved in analgesia as indicated by significantly attenuated Fos-protein induction in the rostral dorsal periaqueductal grey (DPAG) as well as rostral and caudal axes of the ventrolateral PAG (VLPAG). Formalin injections were associated with increased Fos-protein labelling in lateral habenula (LHb) as compared to medial habenula (MHb), however the intensity of this labelling did not differ as a result of neonatal immune challenge. These data highlight the importance of neonatal immune priming in programming inflammatory pain sensitivity later in development and highlight the PAG as a possible mediator of this process.     

Web-Based Software for Rapid Top-Down Proteomic Identification of Protein Biomarkers,with Implications for Bacterial Identification

We have developed web-based software for the rapid identification of protein biomarkers of bacterial microorganisms. Proteins from bacterial cell lysates were ionized by matrix-assisted laser desorption ionization (MALDI), mass isolated, and fragmented using a tandem time of flight (TOF-TOF) mass spectrometer. The sequence-specific fragment ions generated were compared to a database of in silico fragment ions derived from bacterial protein sequences whose molecular weights are the same as the nominal molecular weights of the protein biomarkers. A simple peak-matching and scoring algorithm was developed to compare tandem mass spectrometry (MS-MS) fragment ions to in silico fragment ions. In addition, a probability-based significance-testing algorithm (P value), developed previously by other researchers, was incorporated into the software for the purpose of comparison. The speed and accuracy of the software were tested by identification of 10 protein biomarkers from three Campylobacter strains that had been identified previously by bottom-up proteomics techniques. Protein biomarkers were identified using (i) their peak-matching scores and/or P values from a comparison of MS-MS fragment ions with all possible in silico N and C terminus fragment ions (i.e., ions a, b, b-18, y, y-17, and y-18), (ii) their peak-matching scores and/or P values from a comparison of MS-MS fragment ions to residue-specific in silico fragment ions (i.e., in silico fragment ions resulting from polypeptide backbone fragmentation adjacent to specific residues [aspartic acid, glutamic acid, proline, etc.]), and (iii) fragment ion error analysis, which distinguished the systematic fragment ion error of a correct identification (caused by calibration drift of the second TOF mass analyzer) from the random fragment ion error of an incorrect identification.Food-borne illness is a serious and continuing problem, with an estimated 76 million cases in the United States per year (http://www.cdc.gov). It is often caused by bacteria and viruses that are often ubiquitous in the environment and are difficult to eliminate due to their ability to adapt. In addition to the resulting morbidity, food-borne illness also has enormous societal costs, including losses in worker productivity due to illness, recall of food products determined (or suspected) to be contaminated, etc. Consequently, there is a critical need to develop rapid and sensitive methods for detection and accurate identification of food-borne pathogens.A number of techniques have been developed for detection and identification of food-borne pathogens. A relatively recent technique for bacterial identification involves the use of mass spectrometry (MS). Because of its sensitivity and high specificity, MS has become a popular technique for chemicotaxonomic classification of microorganisms (16, 27). The use of MS in the analysis of microorganisms is a relatively recent application that was dramatically accelerated by the development of two ionization techniques in the late 1980s and early 1990s: electrospray ionization (15) and matrix-assisted laser desorption ionization (MALDI) (24, 37). When coupled with time of flight (TOF) MS, MALDI has been demonstrated to be a powerful tool for “fingerprinting” microorganisms by ionization and detection of proteins from intact bacterial cells or extracts resulting from bacterial cell lysis (1, 2, 3, 8-12, 19, 21, 25, 26, 29, 34, 40, 41, 42). Typically, MALDI-TOF MS “fingerprinting” of microorganisms involves analysis using either pattern recognition or bioinformatic algorithms.Pattern recognition analysis compares MALDI-TOF MS spectra of samples of unknown microorganisms to spectra of known microorganisms. A high degree of similarity between the MS spectrum of an unknown microorganism and an MS spectrum of a known microorganism strongly suggests the identity of the unknown microorganism (22, 39, 43). It should be noted that pattern recognition analysis does not rely on actual identification of the biomarker ion peaks in an MS spectrum. It is the pattern generated by multiple ion peaks that constitutes a microorganism''s “fingerprint.” The actual identities of individual ion peaks are not specified, and the peaks could be peaks for any of a number of possible biological molecules generated by a microorganism, including proteins, nucleic acids, lipids, etc.Microorganism identification by bioinformatic analysis of MALDI-TOF MS data involves using the protein molecular weights (MWs) in bacterial genomic databases to assign biomarker ion peaks in a mass spectrum to specific proteins (4, 5, 32, 33, 45). If a significant number of biomarker ion peaks in a mass spectrum correspond to protein MWs for the open reading frames of a microorganism''s genome, then the microorganism is considered identified. Such an analysis has also incorporated the simplest and most common posttranslational modification (PTM) observed for bacterial proteins, N-terminal methionine cleavage (5). It should be noted, however, that “identification” of a microorganism relies solely on a sufficient number of protein MWs derived from open reading frames of its genome corresponding to the m/z of biomarker ions in a MALDI-TOF MS spectrum. However, the protein MW alone is not sufficient to definitively identify a biomarker ion as a specific protein. Protein biomarkers are considered to be tentatively assigned instead of definitively identified.Analysis of samples containing multiple bacterial organisms presents increased challenges for MALDI-TOF MS when protein MW is the sole criterion for protein biomarker identification. Clearly, it would be advantageous if researchers could obtain more information about a biomarker in addition to its MW. In the case of protein biomarkers, this can be accomplished by enzymatically digesting a protein in solution and analyzing its tryptic peptides by MS (peptide mass mapping) or by tandem MS (MS-MS) (sequence tags) (45). Alternatively, it is possible to fragment mature, intact proteins (without digestion) in the gas phase to obtain sequence-specific and PTM information. This approach is referred to as top-down proteomics. Until recently, top-down proteomics was possible only if Fourier transform ion cyclotron resonance MS involving complicated gas phase ion dissociation techniques was used (6, 23).Although not originally designed for top-down proteomics, recently developed MALDI-tandem TOF (MALDI-TOF-TOF) MS was shown to fragment small or modest-size proteins (5 kDa > molecular mass < 15 kDa) without prior digestion (28). Demirev and coworkers (7) identified Bacillus atrophaeus and Bacillus cereus spores by fragmenting their protein biomarkers using a MALDI tandem mass spectrometer and analyzing the sequence-specific fragment ions generated by comparison to in silico fragment ions derived from protein amino acid sequences from genomic databases. Protein and microorganism identities were determined using a probability-based significance-testing algorithm (P value). The P value algorithm calculates the probability that a protein or microorganism identification occurred randomly. The smaller the P value, the lower the probability that an identification occurred randomly. The data analysis was performed using software developed in house (7).In the current study, web-based software and databases, developed in house at the U.S. Department of Agriculture (USDA), were used to identify 10 protein biomarkers from three pure strains of Campylobacter by sequence-specific fragmentation using a MALDI-TOF-TOF mass spectrometer. Many of the protein biomarkers had been identified previously by bottom-up proteomics techniques (9, 11, 12), which provided an excellent data set to test the accuracy and performance of the algorithms incorporated into the software. MALDI-TOF-TOF MS-MS fragment ions were compared with a database of in silico fragment ions derived from bacterial protein sequences. The sequence-specific MS-MS fragment ions were used to identify a protein and thus the source microorganism. A simple peak-matching mathematical algorithm, incorporated into the software, was used to score and rank protein and microorganism identifications. In addition, the P value algorithm of Demirev and coworkers (7) was also incorporated into the USDA software (available with execution of appropriate control usage agreement) for comparison to the peak-matching algorithm. The peak-matching algorithm correctly identified a protein biomarker among as many as ∼1,400 possible bacterial proteins and gave rankings for protein identification comparable to the rankings obtained by more complicated and computationally intensive P value calculation. We often observed enhancement of the score for correct identification when results for MS-MS fragment ions were compared to results for residue-specific in silico fragment ions compared to non-residue-specific in silico fragment ions. In addition, the correctness of the algorithm''s identification was, in certain cases, further confirmed by fragment ion error analysis which compared random error caused by false matches between MS-MS fragment ions and in silico fragment ions with the systematic error observed for correct matches due to drift in the calibration of the TOF mass analyzer (38).(Portions of this work were presented at the 121st AOAC Conference [13] and at the 55th American Society of Mass Spectrometry Conference [14].)     

Development and analysis of a germline BAC resource for the sea lamprey, a vertebrate that undergoes substantial chromatin diminution

Over the last several years, the sea lamprey (Petromyzon marinus) has grown substantially as a model for understanding the evolutionary fundaments and capacity of vertebrate developmental and genome biology. Recent work on the lamprey genome has resulted in a preliminary assembly of the lamprey genome and led to the realization that nearly all somatic cell lineages undergo extensive programmed rearrangements. Here we describe the development of a bacterial artificial chromosome (BAC) resource for lamprey germline DNA and use sequence information from this resource to probe the subchromosomal structure of the lamprey genome. The arrayed germline BAC library represents ∼10× coverage of the lamprey genome. Analyses of BAC-end sequences reveal that the lamprey genome possesses a high content of repetitive sequences (relative to human), which show strong clustering at the subchromosomal level. This pattern is not unexpected given that the sea lamprey genome is dispersed across a large number of chromosomes (n ∼ 99) and suggests a low-copy DNA targeting strategy for efficiently generating informative paired-BAC-end linkages from highly repetitive genomes. This library therefore represents a new and biologically informed resource for understanding the structure of the lamprey genome and the biology of programmed genome rearrangement.     

Cloned transgenic swine via in vitro production and cryopreservation

It has been notoriously difficult to successfully cryopreserve swine embryos, a task that has been even more difficult for in vitro-produced embryos. The first reproducible method of cryopreserving in vivo-produced swine embryos was after centrifugation and removal of the lipids. Here we report the adaptation of a similar process that permits the cryopreservation of in vitro-produced somatic cell nuclear transfer (SCNT) swine embryos. These embryos develop to the blastocyst stage and survive cryopreservation. Transfer of 163 cryopreserved SCNT embryos to two surrogates produced 10 piglets. Application of this technique may permit national and international movement of cloned transgenic swine embryos, storage until a suitable surrogate is available, or the long-term frozen storage of valuable genetics.     

Towards Miscanthus combustion quality improvement: the role of flowering and senescence

In commercially grown Miscanthus × giganteus, despite imposing a yield penalty, postwinter harvests improve quality criteria for thermal conversion and crop sustainability through remobilization of nutrients to the underground rhizome. We examined 16 Miscanthus genotypes with different flowering and senescence times for variation in N, P, K, moisture, ash, Cl and Si contents, hypothesizing that early flowering and senescence could result in improved biomass quality and/or enable an earlier harvest of biomass (in autumn at peak yield). Ideal crop characteristics at harvest are low N and P to reduce future fertilizer inputs, low K and Cl to reduce corrosion in boilers, low moisture to reduce spoilage and transportation costs, and low Si and ash to reduce slagging and consequent operational downtime. Stems and leaves were harvested during summer, autumn and then the following spring after overwinter ripening. In spring, stem contents of N were 30–60 mg kg^?1, P were 203–1132 mg kg^?1, K were 290–4098 mg kg^?1, Cl were 10–23 mg kg^?1 and moisture were 12–38%. Notably, late senescence resulted in increased N, P, K, Cl, moisture and ash contents, and should therefore be avoided for thermochemical conversion. Flowering and senescence led to overall improved combustion quality, where flowered genotypes tended towards lower P, K, Cl and moisture contents; marginally less, or similar, N, Si and ash contents; and a similar higher heating value, compared to those that had not flowered. Such genotypes could potentially be harvested in the autumn. However, one genotype that did not flower in our trial exhibited sufficiently low N and K content in autumn to meet the ENplus wood pellet standards for those traits, and some of the lowest P, moisture and ash contents in our trial, and is thus a target for future research and breeding.