Preparatory Techniques Research Articles

'All in a box' a concept for optimizing microbiological diagnostic sampling in prosthetic joint infections.

BackgroundAccurate microbial diagnosis is crucial for effective management of prosthetic joint infections. Culturing of multiple intraoperative tissue samples has increased diagnostic accuracy, but new preparatory techniques and molecular methods hold promise of further improvement. The increased complexity of sampling is, however, a tough challenge for surgeons and assistants in the operation theatre, and therefore we devised and tested a new concept of pre-packed boxes with a complete assortment of swabs, vials and additional tools needed in the operating theatre for non-standard samples during a clinical study of prosthetic joint infections.FindingsThe protocol for the clinical study required triplicate samples of joint fluid, periprosthetic tissue, bone tissue, and swabs from the surface of the prosthesis. Separate boxes were prepared for percutaneous joint puncture and surgical revision; the latter included containers for prosthetic components or the entire prosthesis. During a 2-year project period 164 boxes were used by the surgeons, 98 of which contained a complete set of samples. In all, 1508 (89%) of 1685 scheduled samples were received.ConclusionWith this concept a high level of completeness of sample sets was achieved and thus secured a valid basis for evaluation of new diagnostics. Although enthusiasm for the project may have been a contributing factor, the extended project period suggests that the ‘All in a box’ concept is equally applicable in routine clinical settings with standardized but complex diagnostic sampling.

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis. The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture. This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Cervical preparation for first trimester surgical abortion

Preparing the cervix prior to surgical abortion is intended to make the procedure both easier and safer. Options for cervical preparation include osmotic dilators and pharmacologic agents. Many formulations and regimens are available, and recommendations from professional organizations vary for the use of preparatory techniques in women of different ages, parity or gestational age of the pregnancy. To determine whether cervical preparation is necessary in the first trimester, and if so, which preparatory agent is preferred. We searched Cochrane, Popline, Embase, Medline and Lilacs databases for randomised controlled trials investigating the use of cervical preparatory techniques prior to first trimester surgical abortion. In addition, we hand-searched key references and contacted authors to locate unpublished studies or studies not identified in the database searches. Randomised controlled trials investigating any pharmacologic or mechanical method of cervical preparation, with the exception of nitric oxide donors (the subject of another Cochrane review), administered prior to first trimester surgical abortion were included. Outcome measures must have included the amount of cervical dilation achieved, the procedure duration or difficulty, side-effects, patient satisfaction or adverse events to be included in this review. Trials under consideration were evaluated by considering whether inclusion criteria were met as well as methodologic quality. Fifty-one studies were included, resulting in 24 different cervical preparation comparisons. Results are reported as odds ratios (OR) for dichotomous outcomes and weighted mean differences for continuous data. When compared to placebo, misoprostol (400-600 microg given vaginally or sublingually), gemeprost, mifepristone (200 or 600 mg), prostaglandin E and F(2alpha) (2.5 mg administered intracervically) demonstrated larger cervical preparation effects. When misoprostol was compared to gemeprost, misoprostol was more effective in preparing the cervix and was associated with fewer gastrointestinal side-effects. For vaginal administration, administration 2 hours prior was less effective than administration 3 hours prior to the abortion. Compared to oral misoprostol administration, the vaginal route was associated with significantly greater initial cervical dilation and lower rates of side-effects; however, sublingual administration 2-3 hours prior to the procedure demonstrated cervical effects superior to vaginal administration.When misoprostol (600 microg oral or 800 microg vaginal) was compared to mifepristone (200 mg administered 24 hours prior to procedure), misoprostol had inferior cervical preparatory effects. Compared to day-prior laminaria tents, 200 or 400 microg vaginal misoprostol showed no differences in the need for further mechanical dilation or length of the procedure; similarly, the osmotic dilators Lamicel and Dilapan showed no differences in cervical ripening when compared to gemeprost, although gemeprost had cervical effects which were superior to laminaria tents. Older prostaglandin regimens (sulprostone, prostaglandin E(2) andF(2alpha)) were associated with high rates of gastrointestinal side-effects and unplanned pregnancy expulsions. Few studies reported women's satisfaction with cervical preparatory techniques. Modern methods of cervical ripening are generally safe, although efficacy and side-effects between methods vary. Reports of adverse events such as cervical laceration or uterine perforation are uncommon overall in this body of evidence and no published study has investigated whether cervical preparation impacts these rare outcomes. Cervical preparation decreases the length of the abortion procedure; this may become increasingly important with increasing gestational age, as mechanical dilation at later gestational ages takes longer and becomes more difficult. These data do not suggest a gestational age where the benefits of cervical dilation outweigh the side-effects, including pain, that women experience with cervical ripening procedures or the prolongation of the time interval before procedure completion. Mifepristone 200 mg, osmotic dilators and misoprostol, 400microg administered either vaginally or sublingually, are the most effective methods of cervical preparation.

Visualizing the Invisible Nanoscale

How does one know what is the right way to visualize the invisible nanoscale? Practices in the visualization processes are studied by moving about in the fi eld of nanoscience/technology. Nine important aspects of visualization processes have identified. Based on nanoscientists’ responses to a small survey, it is argued that some of these aspects–preparatory techniques–which link up to the experimenter’s and visualization regress, are invisible to nanoscientists. Here tacit rules are followed in the production process of visualizations. This contrasts with presentational aspects where nanoscientists are faced with choices, and have to discuss and defend them. Here it is still impossible to follow tacit rules. These presentational aspects might also be closed down in further development of nanotechnology. The paper asks whether it might be important to keep choices visible.

Scanning electron microscopy and histological examination of human seminal plasma coagulum.

Human seminal plasma coagulum was examined by S.E.M. and light microscopy using various preparatory techniques. Two types of structures were observed; porous and laminated. A consistent finding using all the preparatory techniques was the presence of the majority of the spermatozoa on the surface of the coagulum as opposed to the interior. Those spermatozoa present in the interior of the coagulum are attributed to the invagination of the surface layer. The presence of spermatozoa on the surface of seminal plasma coagulum plays an important role in concentrating spermatozoa in close proximity to the cervical os. This allows spermatozoa to reach cervical mucus rapidly and minimizes the time they must remain in the unfavourable environment of the vagina.

Development of an Analytical Scheme for the Determination of Pyrethroid Pesticides in Composite Diet Samples

Analysis of an individual's total daily food intake may be used to determine aggregate dietary ingestion of given compounds. However, the resulting composite sample represents a complex mixture, and measurement of such can often prove to be difficult. In this work, an analytical scheme was developed for the determination of 12 select pyrethroid pesticides in dietary samples. In the first phase of the study, several cleanup steps were investigated for their effectiveness in removing interferences in samples with a range of fat content (1-10%). Food samples were homogenized in the laboratory, and preparatory techniques were evaluated through recoveries from fortified samples. The selected final procedure consisted of a lyophilization step prior to sample extraction. A sequential 2-fold cleanup procedure of the extract included diatomaceous earth for removal of lipid components followed with a combination of deactivated alumina and C(18) for the simultaneous removal of polar and nonpolar interferences. Recoveries from fortified composite diet samples (10 microg kg(-1)) ranged from 50.2 to 147%. In the second phase of this work, three instrumental techniques [gas chromatography-microelectron capture detection (GC-microECD), GC-quadrupole mass spectrometry (GC-quadrupole-MS), and GC-ion trap-MS/MS] were compared for greatest sensitivity. GC-quadrupole-MS operated in selective ion monitoring (SIM) mode proved to be most sensitive, yielding method detection limits of approximately 1 microg kg(-1). The developed extraction/instrumental scheme was applied to samples collected in an exposure measurement field study. The samples were fortified and analyte recoveries were acceptable (75.9-125%); however, compounds coextracted from the food matrix prevented quantitation of four of the pyrethroid analytes in two of the samples considered.

Microscale analysis of proteins in inner ear tissues and fluids with emphasis on endolymphatic sac, otoconia, and organ of Corti.

Here we describe preparatory techniques adapted for the study of proteins of inner ear tissues and fluids that have allowed us to apply state-of-the-art analytical techniques in spite of the minute size and anatomical complexities of this organ. Illustrative examples address unresolved issues of functional and clinical significance. First, we demonstrate how quick-freezing and freeze drying prevents artifacts that arise from sampling endolymphatic sac (ES) content in the liquid state. This set the stage for the generation of the first protein profile of the ES. Identification of crucial proteins will help elucidate mechanisms of endolymph volume regulation and pathogenesis of Meniere's disease. Second, we show how a unique situation allowed identification of otoconial proteins by mass spectrometric analysis without prior separation and we discuss possible roles for these minor otoconins in otoconial development and prevention of degenerative diseases that affect balance. Finally, we demonstrate techniques for the precise dissection of organ of Corti and its substructures, while preserving their near normal chemical state. We extended an earlier study in which we identified a novel calcium-binding protein by IEF, oncomodulin, localized in the outer hair cells and show here the applicability of prefractionation for the screening of calcium-binding proteins of organ of Corti. These studies demonstrate how advanced preparatory and analytical techniques can be applied to studies of the inner ear.

Revised guides for organ sampling and trimming in rats and mice – Part 3: A joint publication of the RITA )and NACAD )groups

This is the third part of a series of three articles on trimming instructions of rat and mouse protocol organs and tissues in regulatory type toxicity studies, covering the urinary, nervous, musculoskeletal, cardiovascular, and lymphoreticular systems. The article is based on the experience of the European RITA and American NACAD working groups and is an extended revision of trimming guides published in 1995 (BAHNEMANN et al.). The optimum localization for tissue preparation, the sample size, the direction of sectioning and the number of sections to be prepared is described organ by organ. These descriptions are illustrated for each organ by a schematic drawing and/or a macro-photograph showing the plane of section as well as a low magnification of the H&E stained slide demonstrating the optimum "end-product". The objectives of this work, as addressed in detail in the first part (Ruehl-Fehlert et al. 2003), are to standardize tissue sampling and trimming for comparison of historical data obtained from different studies and different laboratories, ensure the presence of all relevant target sites for histopathological evaluation and provide technical advice for preparatory techniques during necropsy, fixation and trimming (Crissman et al. 2004).

Revised guides for organ sampling and trimming in rats and mice – Part 2: A joint publication of the RITA )and NACAD )groups

This is the second part of a series of three articles on trimming instructions of rat and mouse protocol organs and tissues in regulatory type toxicity studies, covering the respiratory, male and female genital, and the endocrine systems. The article is based on the experience of the European RITA and American NACAD working groups and is an extended revision of trimming guides published in 1995 (Bahnemann et al.). The optimum localization for tissue preparation, the sample size, the direction of sectioning and the number of sections to be prepared is described organ by organ. These descriptions are illustrated for each organ by a schematic drawing and/or a macro-photograph showing the plane of section as well as a low magnification of the H&E stained slide demonstrating the optimum "end-product". The objectives of this work, as addressed in detail in the first part (Ruehl-Fehlert et al. 2003), are to standardize tissue sampling and trimming, to improve the comparability of historical data obtained from different studies and different laboratories, ensure the presence of all relevant target sites for histopathological evaluation and provide technical advice for preparatory techniques during necropsy, fixation and trimming. dardize tissue sampling and trimming, to improve the comparability of historical data obtained from different studies and different laboratories, ensure the presence of all relevant target sites for histopathological evaluation and provide technical advice for preparatory techniques during necropsy, fixation and trimming.

Use of a unique freezing technology to cryopreserve ovarian tissue

Use of a unique freezing technology to cryopreserve ovarian tissue

Analysis of false-negative diagnoses on endoscopic brush cytology of biliary and pancreatic duct strictures: the experience at 2 university hospitals.

Endoscopic brush cytology is a valuable technique for the diagnosis of pancreatobiliary malignancy. Despite its widespread use, the sensitivity of this method has been reported as approximately 50%. The specificity is usually higher than 95%. Few reports have systematically analyzed the reasons for this relatively low sensitivity. To determine the rate and reasons for false-negative diagnoses in endoscopic brushing cytology of biliary and pancreatic ducts based on the results of sensitivity, specificity, accuracy, and positive and negative predictive values. Retrospective analysis of laboratory data and slide review of false-negative cases. Two tertiary care state university hospitals. A total of 183 pancreatobiliary brushing specimens obtained from patients undergoing endoscopic retrograde cholangiopancreatography for biliary or pancreatic duct disease for a 4- to 5-year period. Endoscopic retrograde cholangiopancreatography brushings. Determination of sensitivity, specificity, accuracy, and positive and negative predictive values. Analysis of false-negative results. The sensitivity, specificity, accuracy, and positive and negative predictive values, overall, were 48%, 98%, 79%, 92%, and 76%, respectively. Sampling error was a major cause of false-negative diagnoses (67%), followed by interpretive (17%) and technical errors (17%). Improvements in sensitivity and diagnostic accuracy for cancer of the pancreatobiliary tract can be achieved by optimizing slide preparatory techniques. Also, enhancement of the cytologist's diagnostic skills enables the identification of the morphologic features of premalignant lesions. Repeat brushings are indicated for suspicious or negative results not consistent with the clinical or radiologic findings.

Psychologic preparation to decrease anxiety associated with cardiac catheterization.

Undergoing a cardiac catheterization arouses anxiety of most patients. Coronary angiography plays an essential role in the diagnostic evaluation of the patient with suspected or known cardiac disease, and it is an important preoperative cardiac screening protocol for vascular surgery patients. Studies on psychologic preparation have concluded that preparatory techniques--specifically, sensory-perceptual informational and modeling--are beneficial in reducing patients' anxiety in various situations, including cardiac catheterization. The purpose of this study was to compare the preparatory informative (sensory and procedural data) and modeling strategies in reducing anxiety before cardiac catheterization. The research hypothesis that persons (n = 30) given a combination of sensory-perceptual informational preparation and modeling preparation would exhibit less anxiety than persons receiving one preparation alone was not supported. Although no significant difference existed between the postintervention anxiety scores for the 3 different treatment groups, a significant difference existed between the preintervention and postintervention anxiety scores overall. Therefore, this study validates the psychologic and nursing literature that states psychologic preparation is beneficial in reducing patients' anxiety before a stressful experience such as cardiac catheterization. The study findings also revealed an interesting trend for employing a modeling technique. This information contributes to nursing knowledge and may alter and make more effective the nature of standard psychologic preparation for patients undergoing any invasive procedure.

Strategies for preparing clients for treatment. A review.

Substantial numbers of mental health clients do not return following their initial therapy visits or drop out of treatment prematurely. Two general classes of strategies designed to reduce premature attrition and enhance treatment participation are reviewed. Research on psychotherapy preparatory techniques (role induction, vicarious therapy pretraining, and experiential pretraining) indicates that these educational techniques are effective in reducing early treatment attrition and may be especially effective with populations at high risk for dropout (e.g., lower socioeconomic groups, chronically mentally ill clients, and institutionalized juvenile delinquents). Motivational interviewing, a technique originally developed for clients with alcohol problems, is designed to reduce client ambivalence toward therapy and change and enhance commitment to and motivation for treatment. Research in the alcohol field suggests that a session of pretreatment motivational interviewing enhances treatment outcome. Both motivational interviewing and psychotherapy preparatory techniques are relatively brief and easy to incorporate into existing mental health care.

Suturing.

This article describes the nursing interventions involved in wound cleaning before the application of sutures, as well as the necessary wound assessment skills, and preparatory techniques in suturing simple lacerations of the skin.

Assessment of Vaginal Lactobacillary Flora in Wet Mount and Fresh or Delayed Gram's Stain

Objective: The assessment of the vaginal lactobacillary flora helps to direct further diagnostic microbiologic investigations in genital infectious disease and seems to represent a powerful tool in predicting infectious morbidity and preterm labor during pregnancy. In the absence of a “gold standard,” we studied the variations in assessing lactobacillary morphotypes according to the method used. Methods: The lactobacillary flora from 183 pregnant women was classified according to 3 groups: normal, intermediate, and abnormal. This grading of lactobacilli was appled to vaginal and cervical specimens by means of 1) immediate wet-smear microscopy, 2) Gram's stain on a fresh, air-dried specimen, and 3)delayed Gram's stain after specimen transportation in Stuart's growth medium for 3–6 h. Results: The assignment of intermediate or abnormal flora (grade II or grade III) showed high concordance rates among the different preparatory techniques, but the assignment of grade I (normal flora) did not. Fewer lactobacilli were found 2.6 times more often after Gram's stains of fresh specimens [Relative Risk (RR) 2.6, 95% confidence interval (CI) 1.7–4.1] and 6 times more often when the Gram%s stain was performed in a delayed examination after transport than in a fresh wet-mount specimen (RR 6.2, 95% CI 2.5–15.6). Disturbed lactobacillary grades were also found more frequently in specimens from the cervix than those from the vagina (RR 4.0, 95% CI, 1.5–10.4). Conclusions: There are discrepancies in the diagnosis of lactobacillary grades between gram-stained and fresh vaginal specimens. The evidence is ambiguous as to which of the 2 methods is responsible. If an evaluation is to be done on a gram-stained specimen, then the storage of the sample in Stuart transport medium before staining should be avoided.

Assessment of vaginal lactobacillary flora in wet mount and fresh or delayed Gram's stain

Objective The assessment of the vaginal lactobacillary flora helps to direct further diagnostic microbiologic investigations in genital infectious disease and seems to represent a powerful tool in predicting infectious morbidity and preterm labor during pregnancy. In the absence of a “gold standard,” we studied the variations in assessing lactobacillary morphotypes according to the method used. Methods The lactobacillary flora from 183 pregnant women was classified according to 3 groups: normal, intermediate, and abnormal. This grading of lactobacilli was appled to vaginal and cervical specimens by means of 1) immediate wet-smear microscopy, 2) Gram's stain on a fresh, air-dried specimen, and 3) delayed Gram's stain after specimen transportation in Stuart's growth medium for 3–6 h. Results The assignment of intermediate or abnormal flora (grade II or grade III) showed high concordance rates among the different preparatory techniques, but the assignment of grade I (normal flora) did not. Fewer lactobacilli were found 2.6 times more often after Gram's stains of fresh specimens [Relative Risk (RR) 2.6, 95% confidence interval (CI) 1.7–4.1] and 6 times more often when the Gram's stain was performed in a delayed examination after transport than in a fresh wet-mount specimen (RR 6.2, 95% CI 2.5–15.6). Disturbed lactobacillary grades were also found more frequently in specimens from the cervix than those from the vagina (RR 4.0, 95% (CI, 1.5–10.4). Conclusions There are discrepancies in the diagnosis of lactobacillary grades between gram-stained and fresh vaginal specimens. The evidence is ambiguous as to which of the 2 methods is responsible. If an evaluation is to be done on a gram-stained specimen, then the storage of the sample in Stuart transport medium before staining should be avoided. © 1996 Wiley-Liss, Inc.

Programming the microwave oven

Microwaves can be used to stimulate chemical bonding, diffusion of reagents into and out of the specimen, and coagulation processes in preparatory techniques. Temperature plays an important role in these processes. There are several ways of controlling the temperature of microwave-exposed tissue, fluids and solids that are used in these preparatory techniques. In this paper we describe our accumulated experience with monitoring and controlling microwave-oven parameters with the help of a personal computer (PC). A specially designed microwave oven was used with an advanced temperature probe. The oven was connected to an IBM-compatible PC. This enabled us to analyze the quality of temperature control, and the relationship between displayed and actual temperature. We show that for microwave-transparent materials the addition of a microwave-absorbent plate to the material results in better temperature control.

Localisation of bone‐forming cells during fracture healing by osteocalcin immunocytochemistry: An experimental study of the rabbit tibia

An immunocytochemical method was used to localise osteocalcin-producing cells during fracture healing in a rabbit model. In preliminary studies, tibial growth plates from young rabbits were used as a source of new bone formation, in order to determine the optimal tissue preparatory techniques. In the present study, a tibial shaft fracture was created in adult rabbits to study closed fracture healing. An indirect peroxidase method was used to stain paraffin-embedded tissue sections for osteocalcin. Osteocalcin-producing cells were positively identified at the periosteal and endosteal surfaces near the fracture site. Osteocalcin staining was not demonstrated in the surrounding soft tissues. At the interface between newly formed bone trabeculae and the cartilage layer within the callus, chondrocytic cells consistently showed localisation of osteocalcin. Within cartilaginous areas of the callus, some chondrocytes showed positive staining for osteocalcin. These cells were often seen in the proximity of blood vessels. The findings suggest that during fracture healing, under certain conditions, chondrocytes are capable of producing osteocalcin and thus could be considered capable of possible transformation into osteoblasts.

The problem of hot spots in microwave equipment used for preparatory techniques—Theory and practice

AbstractElectron microscopists who wants to use a microwave (MW) oven to stimulate preparatory processes are sooner or later confronted with the problem of hot spots. It soon becomes clear to the user of any MW oven that the energy distribution—thus the speed of absorbing energy, and hence warming up—varies topographically. The unaware can observe variations in results when the principles of topographic order are not followed meticulously. To understand the hot‐spot phenomenon a certain theoretical knowledge is needed, as presented in this paper. In the 10 years that we have used MW ovens for our preparatory techniques we have learned how to solve the problem of hot spots. Thermographic paint can be applied to record energy distribution. In our studies, we have used a fiberoptic thermometer to follow precisely in time the temperature in the MW‐exposed media, and these observations have provided us with the needed insight to obtain reproducible results. We argue that only when temperature curves are given by the authors can their recipes be used successfully in other laboratories.

Size, morphology and composition of particulates in aquatic ecosystems: solving speciation problems by correlative electron microscopy.

Particulates can impact directly on aquatic ecosystems by determining the availability and mode of dispersion of both contaminants and nutrients. An understanding of the mechanisms of such particle-associated phenomena is being augmented by particle analysis technology. In this context, microscopic and spectroscopic techniques, devised for problem solving, are being applied to frequently encountered sub-micrometre particulates which are 'unstable' with respect to methods of sample preparation and storage used routinely for particulates prior to analysis. These unstable aquatic particulates include 'species' sensitive to dehydration and to artificial aggregation induced by surfaces within a fractionation apparatus. These species, as defined broadly, include polysaccharide gels, hydrated humic substances, iron oxyhydroxides, viruses, the smallest micro-organisms and decomposing parts of cells. To develop predictive models of their roles as dispersing agents for contaminants, and to speciate such associations, it is necessary to characterize them in a state as close to the natural as possible. This critical review presents the state-of-the-art in the realistic characterization of hydrated sub-micrometre particulates by correlative electron microscopy (EM) used in conjunction with spectroscopy and minimally perturbing preparatory techniques. Correlative EM is a strategy for using several different kinds of microscopes and accessory techniques in a multi-method context to analyse a given specimen for different kinds of information, including relationships in three dimensions within colloid systems. Sizing, morphology and gross composition are determined on a 'per particle' basis by transmission EM used in conjunction with energy-dispersive spectroscopy, electron diffraction and molecule-specific stains.(ABSTRACT TRUNCATED AT 250 WORDS)