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PubMedDate not supplied

Optimising cryopreservation protocols for haematopoietic progenitor cells: a methodological approach for umbilical cord blood.

Current cryopreservation protocols for haematopoietic cells have developed largely empirically and there is no consensus on an optimal method of preservation. These protocols, though providing sufficient cells to permit engraftment, can lead to cell loss of the order of 50 percent. In the context of umbilical cord blood such losses are unacceptable. Whilst an empirical approach can provide an acceptable level of recovery, the cryopreservation process can only be optimised by adopting a methodological approach. This paper provides an overview of just such an approach as illustrated by a study on CD34 cells from umbilical cord blood. It involves firstly the determination of membrane permeability parameters that can then be used to model safe addition and elution protocols for the chosen cryoprotectant, in this case dimethyl sulphoxide. This in turn permits cryoprotectant toxicity to be evaluated free from the confounding effect of osmotic damage caused by inappropriate addition and elution protocols. Finally, non-toxic concentrations of cryoprotectant may be investigated in a cooling rate study to provide an optimal cryopreservation protocol. Using the model, the effect on CD34 cells of current addition and elution protocols was also examined.

Antigens, CD34↗
PubMed1995

[Adequate neuropsychological evaluation of dementia].

The authors discuss the protocol of the Optimal Neuropsychological Evaluation of Dementias of Montreal and its possible application in Brazil. This protocol is important for evaluation in neuropsychological evolution of dementia of the Alzheimer type and for bringing to the fore distinct affected cognitive profiles as much on a transversal base as longitudinal. The authors believe that its application would contribute to the progress of research in neurology, as in the clear distinction between normal aging and pathological aging.

Alzheimer Disease↗
PubMed1992

Multidisciplinary protocol for determining aminoglycoside dosage.

A protocol for determining optimal dosages of aminoglycosides early in therapy is described, and the effectiveness of the protocol is evaluated. The protocol was developed jointly by physicians and pharmacists at a 550-bed hospital to ensure that surgical patients prescribed aminoglycosides were quickly and consistently put on a safe and effective course of therapy. Physicians select an aminoglycoside and calculate a loading dose and initial maintenance dosage by using a nomogram printed on an antimicrobial order form. Nurses are trained to administer and document aminoglycoside doses accurately and to draw blood samples at the correct times. Pharmacists order serum aminoglycoside concentration assays, analyze the results, and recommend changes in dosage when necessary. To evaluate the effectiveness of the dosing protocol, the records of surgical patients treated before and after the protocol was in place were reviewed. Compared with the control group, a higher percentage of patients treated under the protocol were receiving therapeutic, nontoxic dosages of aminoglycosides within 48 hours of the start of therapy. In addition, fewer serum drug concentration tests were ordered per patient under the protocol, and the percentage of concentration determinations useful for analysis was higher. The mean duration of aminoglycoside therapy was identical before and after the protocol was instituted, and nephrotoxic reactions tended to be less common among the protocol patients. An aminoglycoside dosing protocol requiring the cooperation of pharmacists, nurses, and physicians provides a consistent, safe, and effective means of managing aminoglycoside therapy for the hospitalized patient.

Adult↗
PubMedDate not supplied

A prospective, randomized clinical trial comparing two hyperbaric treatment protocols for carbon monoxide poisoning.

INTRODUCTION: The optimal hyperbaric oxygen (HBO2) treatment protocol for acute carbon monoxide (CO) poisoning is unknown. This is indicated by one study that found 18 different protocols to treat CO poisoning by North American multiplace hyperbaric facilities. A pilot study was conducted to evaluate the feasibility of randomizing patients to different protocols and to determine whether any large differences in clinical outcome were present between the two most common protocols. METHODS: Adult patients with accidental CO poisoning resulting in transient loss of consciousness, presentation to the emergency department within 12 hours, primary language English, high school education, and residence within 100 miles of the hyperbaric facility were recruited. Enrolled patients were randomized to one HBO2 treatment at 2.4 atmospheres absolute (atm abs) pressure with 90 minutes of 100% oxygen breathing vs. treatment by the US Air Force CO protocol (3.0 atm abs maximum pressure). A neurocognitive screening test was performed immediately after hyperbaric treatment and repeated 14-21 days later. RESULTS: From 1995 to 2002, 30 patients age 21 to 88 years were randomized, 18 to treatment at 2.4 atm abs and 12 to 3.0 atm abs. Average carboxyhemoglobin level for the population was 24.8 +/- 8.8% (mean +/- SD). Delay to hyperbaric treatment averaged 313 +/- 129 minutes. Neither variable was different between treatment groups. Six patients had abnormal neurocognitive testing immediately following hyperbaric treatment, 4 in the 2.4 atm abs group (22%) and 2 in the 3.0 atm abs group (17%) (P=0.71). One patient in each group demonstrated abnormality on delayed testing (p=0.75). One in each group did not return for follow-up. CONCLUSIONS: It is feasible to randomize CO-poisoned patients to different hyperbaric treatment protocols. Determination of differences in efficacy between treatment protocols will require a large multicenter trial with the use of detailed neurocognitive testing.

Adult↗
PubMed1997

Collapsed ultraendurance athlete: proposed mechanisms and an approach to management.

OBJECTIVE: To review the common conditions causing collapse in ultraendurance athletes, to propose appropriate treatment protocols for the more common medical conditions that are encountered, and to provide practical guidelines for the management of the medical facilities at these endurance events. DATA SOURCES: Books published on the subject, abstracts of the American College of Sports Medicine Annual Congress dealing with the subject over the last 5 years, and electronic search of the Medline and Current Contents databases (last searched May 1995). German articles were included in the search criteria. STUDY SELECTION: Articles dealing generally with the management of medical facilities at endurance events were chosen. Articles dealing more specifically with the common medical conditions encountered at these events were then sourced and included in this review. DATA EXTRACTION: Multiple reader extraction of relevant data. DATA SYNTHESIS: A practical guideline to the management of the medical facility at an endurance event is detailed, expanding specifically on the nature of the conditions causing collapse during or after endurance exercise with a proposed classification of causes of collapse for optimizing immediate management and management protocols for specific conditions such as heatstroke, hyponatremia, hypoglycemia, exercise-associated collapse, and muscle cramps. CONCLUSION: The most commonly encountered medical condition at endurance athletic events is collapse after the event. The popular view that all persons who collapse have dehydration-induced hyperthermia has been challenged. Here we extend that argument and provide diagnostic and management protocols and propose a triage system that considers the most common causes of collapse in ultraendurance athletes. These protocols can optimize the efficient and safe management of large numbers of collapsed ultraendurance athletes. It is proposed that these protocols can optimize the efficient and safe management of large numbers of collapsed ultra endurance athletes.

Adult↗
PubMed2001

Assessment of mitochondrial membrane potential in yeast cell populations by flow cytometry.

In yeast the use of rhodamine 123 (Rh123) has been restricted to the evaluation of mitochondrial respiratory function including the discrimination between respiratory-competent and -deficient cells. This study describes the optimization and validation of a low-concentration Rh123 staining protocol for the flow-cytometric assessment of mitochondrial membrane potential (Delta Psi m) changes in whole yeast cells. The optimized protocol was validated by the use of compounds that specifically affect mitochondrial energetics. Epifluorescence microscopy was used to monitor Rh123 distribution within the cell. Incubation of yeast cell suspensions with Rh123 (50 nM, 10 min) gave minimal non-specific binding and cytotoxicity of the dye. The ratio (R) between the green fluorescence and forward scatter (both measured as log values) was used to measure Delta Psi m with only little dependence on cell 'volume' and mitochondrial concentration. Cells treated with mitochondrial membrane de- or hyper-polarizing agents displayed a decrease and an increase of R values respectively, indicating that changes of the Rh123 distribution in cells indicate variations in the Delta Psi m. Live and dead cells also displayed significantly different R values. The method described here allows assessment of Delta Psi m changes in whole yeast cells in response to a given drug. Moreover, the relationship between drug effects and disorders of mitochondrial energetics might be addressed.

Flow Cytometry↗
PubMed2004

Multi-detector row CT: principles and practice for abdominal applications.

Abdominal imaging with multi-detector row computed tomography (CT) can be performed during short breath holds. On 16-channel multi-detector row CT scanners, the effective detector row thickness, depending on the manufacturer, is typically 1.0-1.5 mm, which results in a beam collimation of 16-24 mm. At a gantry rotation speed of 0.5 second and a pitch of 1, the table travel speed will be 32-48 mm/sec. At a smaller effective detector row thickness and a narrower beam collimation, a slightly higher pitch may be needed to obtain short-breath-hold CT scans. Typically, transverse scans are viewed at a reconstructed section thickness of 3-5 mm, with thinner sections used for CT angiography and whenever off-axial reformations are obtained. The radiologic technique should be optimized according to the transverse section thickness used for interpretation, and the contrast material administration protocol should be optimized according to the clinical problem, with the scanning triggered for enhancement of a target organ.

Contrast Media↗
PubMed2005

Simultaneous population optimal design for pharmacokinetic-pharmacodynamic experiments.

Multiple outputs or measurement types are commonly gathered in biological experiments. Often, these experiments are expensive (such as clinical drug trials) or require careful design to achieve the desired information content. Optimal experimental design protocols could help alleviate the cost and increase the accuracy of these experiments. In general, optimal design techniques ignore between-individual variability, but even work that incorporates it (population optimal design) has treated simultaneous multiple output experiments separately by computing the optimal design sequentially, first finding the optimal design for one output (eg, a pharmacokinetic [PK] measurement) and then determining the design for the second output (eg, a pharmacodynamic [PD] measurement). Theoretically, this procedure can lead to biased and imprecise results when the second model parameters are also included in the first model (as in PK-PD models). We present methods and tools for simultaneous population D-optimal experimental designs, which simultaneously compute the design of multiple output experiments, allowing for correlation between model parameters. We then apply these methods to simulated PK-PD experiments. We compare the new simultaneous designs to sequential designs that first compute the PK design, fix the PK parameters, and then compute the PD design in an experiment. We find that both population designs yield similar results in designs for low sample number experiments, with simultaneous designs being possibly superior in situations in which the number of samples is unevenly distributed between outputs. Simultaneous population D-optimality is a potentially useful tool in the emerging field of experimental design.

Computer Simulation↗
PubMed2005

Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations.

Antibodies are valuable tools in the laboratory and clinic. Antibodies include those secreted by a single clone of B lymphocytes, termed monoclonal antibodies, and those produced by a mixture of various B lymphocyte clones, termed polyclonal antibodies. Both products have become essential instruments in fundamental immunological research, immunohistochemistry, diagnostic testing, and vaccine quality control. Antibody production requires a substantial number of animals, and the animals are subjected to a number of invasive procedures such as antigen injection and blood collection. However, by carefully designing an immunization protocol and by optimizing the immunization response, it is possible to minimize animals' pain and distress while obtaining optimal immune responses. In this article, the critical steps in the production of polyclonal and monoclonal antibodies are described, specifically including selection of the animal species and its age, injection protocol, and ascites tapping. Recommendations are provided for optimizing the immunization response.

Adjuvants, Immunologic↗
PubMed2006

Protocol modifications influence the result of EGF receptor immunodetection by EGFR pharmDx in paraffin-embedded cancer tissues.

EGF receptor (EGFR) became a useful target for several recently introduced therapies of various cancer types including colorectal, lung, head and neck cancers and glioblastoma. The successful clinical application of these novel molecularly targeted therapies requires the expression of their target, EGFR, determined by nucleic acid based or immunohistochemical techniques. However, until now, immunohistochemistry has not become a reliable diagnostic approach for this purpose. The golden standard for the determination of EGFR protein expression in paraffin-embedded cancer tissues is the EGFR pharmDxTM kit. Here we show that the recommended protocol may not be optimal for EGFR immunodetection. Microwave antigen retrieval and extended primary antibody incubation time converted four out of eight EGFR-negative tumors into EGFR-positive in a study of 50 lung adenocarcinoma cases. Accordingly, we recommend retesting cases negative for EGFR with EGFR pharmDxTM using protocol modifications optimizing antigen retrieval and the incubation periods.

Adenocarcinoma↗
PubMed1986

Hepatic gluconeogenesis from alanine: 13C nuclear magnetic resonance methodology for in vivo studies.

This paper describes an experimental protocol designed to optimize 13C NMR spectra from the liver of the living anesthetized rat at 1.9 T. The protocol involves the use of a Helmholtz NMR coil which is positioned around the liver after surgical exposure. 1H decoupling is facilitated by double tuning this coil to both the 1H and the 13C frequencies. The protocol was shown to be suitable for studying the hepatic metabolism of 13C-labeled substrates in vivo by investigating the metabolism of [3-13C]alanine. Labeled glucose, glutamate, glutamine, and aspartate were formed and detected by 13C NMR in vivo in this experiment. The labeling patterns in these metabolites provided evidence that the major flow of alanine carbon into the Krebs cycle is via the pyruvate carboxylase reaction rather than through pyruvate dehydrogenase.

Alanine↗
PubMed1992

Development of immunotherapeutic strategies for the treatment of malignant neoplasms.

Focused preclinical studies have been used to gain insight into the mechanism of therapeutic activity of cytokines, growth factors and biological response modifiers (BRMs). These data can then be used to develop a clinical hypothesis to facilitate the development of these new biological drugs. In this manuscript, we discuss a number of preclinical and clinical studies using interferon-gamma, IL-2, and the colony stimulating factors. The importance of the systematic profiling of the biological activity of such biological drugs is emphasized and we discuss the utility of the mechanistic data in their clinical development. The overall preclinical approach identifies the cellular, biochemical or gene regulatory event that is associated with the therapeutic activity of a biologic and this surrogate (be it biological, chemical, or quality of life) is then used to optimize the clinical protocol in a phase 1b trial. This, in theory, results in the rapid identification of the optimal dose, schedule and route of administration for subsequent testing in a phase II/III clinical trial.

Animals↗
PubMed2003

Extraction of high-quality genomic DNA from latex-containing plants.

The isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including long PCR, endonuclease restriction digestion, Southern blot analysis, and genomic library construction. Many protocols are available for the extraction of DNA from plant material. However, for latex-containing Asteraceae (Cichorioideae) species, standard protocols and commercially available kits do not produce efficient yields of high-quality amplifiable DNA. A cetyltrimethylammonium bromide protocol has been optimized for isolation of genomic DNA from latex-containing plants. Key steps in the modified protocol are the use of etiolated leaf tissue for extraction and an overnight 25 degrees C isopropanol precipitation step. The purified DNA has excellent spectral qualities, is efficiently digested by restriction endonucleases, and is suitable for long-fragment PCR amplification.

Asteraceae↗
PubMed1994

A study of simulated annealing protocols for use with molecular dynamics in protein structure prediction.

The method of simulated annealing can be of use in protein structure prediction by homology modelling where side chain conformations must be predicted. In this study an attempt has been made to optimize a molecular dynamics method for this purpose. Heating and cooling protocols to maximize the accuracy of the predictions have been developed. The optimized protocol involves cooling from 3000 to 0 K over 20 ps while simultaneously introducing the non-bonded energy term. The use of a 'soft' non-bonded interaction energy term in place of a standard 6-12 potential is found to be important. The reliability of the predictions has been analysed in terms of the environment of the residues (solvent accessibility) and the degree of uncertainty in the structure (number of unknown torsion angles). Depending on these factors the percentage of unknown side chain torsion angles that are correctly predicted within 30 degrees ranges from approximately 50 to 75%. Potential problems and limitations of the method are discussed.

Amino Acid Sequence↗
PubMedDate not supplied

High-throughput DNA preparation system.

A system demonstrating the feasibility of high-throughput, centrifugation-based DNA separations and purifications has been constructed and tested. Samples are currently processed at a rate of 96 in approximately 2-3 h. The device implements an automation-optimized alkaline lysis protocol for the rapid extraction of plasmid or cosmid DNA from 1-ml bacteria cultures. The conditions for optimal culturing in deep-well (96 x 1 ml) microwell plates have been developed, and all sample manipulations are done within these plates. The use of microwell plates was essential to obtain high throughput and make manipulations following the DNA preparation (prep) easier because they can then be manipulated using a variety of commercially available robots. The entire prep system is constructed above a Beckman GPR centrifuge and operated under Macintosh IIcx control. This device has systems for fluid handling, microwell-plate manipulations, and centrifuge rotor alignment.

Automation↗
PubMed2006

ABO-incompatible solid-organ transplantation.

To increase transplantation access, particularly in living-donor renal transplantation, efforts have been made to overcome the barrier of ABO incompatibility. In adults, the most successful cases have involved renal transplantation. Although the overall goal of reducing antibodies against donor ABO before and after transplantation is a general principle, the protocols used to accomplish this goal vary. More well-designed, controlled clinical trials are needed to establish optimal peritransplantation management protocols. Incompatible liver transplantation still is viewed as a temporary measure until ABO-compatible transplantation can be performed. ABO-incompatible heart and lung transplantation in adults still is not performed intentionally. In children, particularly those with relatively immature immune systems, ABO-incompatible transplantation generally has more success. The immunologic mechanisms leading to successful transplantation are being elucidated. Accommodation is a process whereby the donor organ may participate in its own survival through a series of protective gene responses, possibly in response to low-level incompatible antibody (HLA and ABO). In infants, spontaneous, acquired B-cell tolerance seems to be a primary mechanism. Peritransplantation therapy might be tailored to invoke specific immune graft-sparing mechanisms. The stage is set to eliminate ABO as a barrier to solid-organ transplantation.

ABO Blood-Group System↗
PubMed2003

Optimizing stringency for expression microarrays.

While several studies have reported methods to optimize expression microarray protocols, none have dealt directly with hybridization wash stringency. We designed a series of experiments to determine the optimal stringency conditions for microarray experiments, using reproducibility and magnitudes of log2 (test/reference) ratio values as measures of quality. Low-stringency wash conditions of cell line hybridizations led to nonspecific binding, resulting in increased intensities, decreased magnitude of ratios, and poor reproducibility. Relatively high-stringency wash conditions were found to give the best reproducibility and large magnitude ratio changes, although increasing the stringency beyond this point led to lower magnitude ratios and poorer reproducibility. The expression levels of the ERBB2 oncogene in the BT474 versus MCF7 cell lines showed that high-stringency wash conditions gave the best agreement with real-time quantitative PCR, although the magnitude of the changes by microarray was smaller than for real-time quantitative PCR. Analysis of a series of cell lines washed at the optimized stringency indicated that the rank order of relative expression levels for ERBB2 microarray clones agreed well with the rank order of ERBB2 levels, as measured by quantitative PCR. These results indicate that the optimization of stringency conditions will improve microarray reproducibility and give more representative expression values.

Artifacts↗
PubMedDate not supplied

Left ventricular assist device weaning: hemodynamic response and relationship to stroke volume and rate reduction protocols.

Clinical evidence of myocardial recovery in a small cohort of patients supported with a left ventricular assist device (LVAD) has been reported. Development of an optimal LVAD weaning protocol is needed for these patients to sustain recovery after device explant. In this study, we tested the hypothesis that LVAD stroke volume reduction produces a steady-state mechanical reloading of left ventricular (LV) pressures and volumes compared with LVAD rate reduction that results in transient mechanical reloading of the heart due to beat-to-beat variation in LV pressures and volumes. The relationship of LVAD flow to LVAD stroke volume and systolic interval over a range of LVAD rates (60, 80, 100, 120, and 140 bpm) was validated in a mock circulatory flow loop. In six acute experiments, calves were implanted with a pneumatic paracorporeal LVAD (PVAD, Thoratec, Pleasanton, CA). The PVAD was operated asynchronously in the auto volume mode (full decompression) for 30 minutes to establish a baseline control condition. The calf hearts were then mechanically reloaded by LVAD rate reduction (80, 60, and 40 bpm) or LVAD stroke volume reduction (100, 120, and 140 bpm) protocols consisting of 30 minutes of support at each LVAD beat rate. The order of weaning protocols was randomized with a 30-minute recovery period (LVAD volume mode to fully decompress heart allowing it to rest) between protocols to enable return to baseline control state. Aortic pressure and flow, LV pressure and volume, pulmonary artery flow, and LVAD flow waveforms were recorded for each test condition. The LVAD stroke volume reduction protocol produced steady-state mechanical reloading compared with VAD rate reduction that resulted in transient LV mechanical reloading. This distinction is due to differences in their temporal relationships between LVAD and LV filling and emptying cycles. The acute hemodynamic benefit of LVAD stroke volume reduction was greater reduction in LV end-diastolic pressure and increase in LV segmental shortening than LVAD rate reduction. The long-term effects of steady-state and transient LV mechanical reloading on myocardial structure and function toward achieving sustained myocardial recovery warrant further investigation.

Animals↗