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Results for “protocol optimization”

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PubMed2003

Determining and optimizing the precision of quantitative measurements of perfusion from dynamic contrast enhanced MRI.

PURPOSE: To examine the sensitivity of quantitative dynamic contrast enhanced MRI (DCE-MRI) perfusion maps to errors in the various source images and to determine optimal imaging parameters for reducing this sensitivity. MATERIALS AND METHODS: A detailed analysis of the precision of a DCE-MRI protocol was performed using the "propagation of errors" technique to investigate the effect of errors in the source images on errors in K(trans). Optimal parameter values and interactions between parameters were examined. The propagation of errors analysis was validated by Monte-Carlo simulations. RESULTS: The precision of K(trans) was found to be most sensitive to artifacts in the tissue portion of the baseline images and least sensitive to noise in the arterial portion of the dynamic images. The tip-angle strongly affected the precision, with the optimum being a function of tissue T1(0). CONCLUSION: Protocol optimization requires matching the tip-angle to the anticipated T1(0) of the tissue of interest; however such optimization yields a relatively small improvement. Future developmental efforts would be most productively focused on minimizing the artifact level.

Artifacts↗
PubMed1993

Chemotherapy outcomes in advanced non-small-cell lung carcinoma.

Despite 20 years of chemotherapy trials in advanced NSCLC, optimal regimens leading to complete remissions have not been identified. The decision to treat a patient who has inoperable advanced NSCLC must take into account the toxicity of the chemotherapy. The toxicities most often reported are myelosuppression and emesis; however, these trials were performed before the use of colony-stimulating factors (ie, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor) and serotonin antagonists (ie, ondansetron). Granulocyte colony-stimulating factor has been shown to shorten the duration of neutropenia and thus decrease the incidence of confirmed infections. Colony-stimulating factors also may allow significant escalation of the dose of chemotherapy. Ondansetron has been shown to ameliorate cisplatin-induced emesis better than other antiemetics. The performance status of a patient has been noted to be a predictor for survival, as well as response to therapy, and this should also be taken into consideration when deciding to treat a patient with advanced inoperable NSCLC. Ideally, patients with stage IV NSCLC should be placed on investigational therapy protocols to identify optimally active combinations of agents. One approach to the patient with inoperable NSCLC who is ineligible for a trial, or who does not wish to participate in a trial, is to offer chemotherapy soon after diagnosis, as patients in this category are likely to be less symptomatic and have optimal performance status. A platinum-containing regimen would seem to be the most reasonable regimen in such a patient. It is hoped that ongoing trials in suitable candidates will lead to the identification of more consistently active agents to deal with this devastating disease.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2006

A fast and reliable route integrating calibration and analysis protocols for water-soluble vitamin determination on microchip-electrochemistry platforms.

A novel analytical route to determine water-soluble vitamins (B group and C) using single channel microchip-electrochemistry platforms is presented. The electrochemical detection protocol was carefully optimized, and it was shown that it was crucial to use 1 M nitric acid in the detector compartment to detect folic acid. A phosphate buffer (pH 6, 10 mM) and a separation voltage of 2 kV gave the complete separation of vitamins in less than 130 s, with good reproducibility (RSDs less than 10%) and accuracy (error less than 9%). In addition, a methodological innovation integrating calibration and analysis of water-soluble vitamins on the chip is also proposed. The strategy consisted in sequentially using both reservoirs (named calibration and analysis reservoirs) as well as a calibration factor (defined as signal/concentration of analyte). The analytical route required 350 s in the overall protocol (employing 130 s in calibration plus 130 s in analysis), an improvement over the times used in both conventional and microchip protocols.

Ascorbic Acid↗
PubMed2006

CINE-MR imaging of the normal and infarcted rat heart using an 11.7 T vertical bore MR system.

MR imaging is uniquely placed to non-invasively study rodent cardiac structure and function. High-field MR scanners commonly have a vertical bore, and the purpose of this work was to demonstrate CINE-MR imaging in normal and infarcted rat hearts after determining hemodynamic stability when positioned vertically for imaging. Optimisation of imaging parameters was carried out prior to assessment of cardiac function in a group of normal and infarcted rat hearts. Rat hemodynamics were unaltered when vertical for 90 minutes, compared with horizontal measurements and rat cardiac parameters were measured accurately and reproducibly with our optimized CINE-MR protocol. A flip angle of 17.5 degrees was shown to provide optimal contrast for the assessment of structure and function, and, in contrast to our findings in mice, respiratory gating was not found to be essential. Hence, we conclude that vertical bore MR systems can be used to measure in vivo cardiac function in normal and infarcted rat hearts.

Analysis of Variance↗
PubMed1989

Synthesis and characterization of mu-conotoxin IIIa.

mu-Conotoxin IIIa, a voltage-dependent sodium channel neurotoxin, has been synthesised using solid-phase peptide synthesis employing 9-fluorenylmethoxycarbonyl chemistry. After cleavage from the resin, the peptide was isolated by reverse-phase HPLC and then the six acetamidomethyl groups were removed by treatment with mercuric acetate. The reduced product so formed was purified by reverse-phase HPLC. Protocols were developed to optimize the oxidation of the cysteine residues to form disulphide bonds. Protocols employed using air oxidation together with 2-mercaptoethanol were the most effective. As complete oxidation was never obtained the oxidised peptide was purified by reverse-phase HPLC. The activity of our products was monitored using [3H]saxitoxin binding to eel membranes. The oxidised product was able to completely block [3H]saxitoxin binding in a competitive manner. Lineweaver-Burke analysis of [3H]saxitoxin binding gave a Ki of 1.5 nM, IC50 was determined as 26.6 nM. It was also shown that the pure synthetic mu-conotoxin IIIa had the same retention time on reversephase HPLC as the natural conotoxin IIIa. Thus an active toxin has been synthesised that can be used to probe sodium channels.

Binding Sites↗
PubMed2006

Threshold feedback control for a collective flashing ratchet: threshold dependence.

We study the threshold control protocol for a collective flashing ratchet. In particular, we analyze the dependence of the current on the values of the thresholds. We have found analytical expressions for the small threshold dependence both for the few and for the many particle cases. For few particles the current is a decreasing function of the thresholds, thus, the maximum current is reached for zero thresholds. In contrast, for many particles the optimal thresholds have a nonzero finite value. We have numerically checked the relation that allows to obtain the optimal thresholds for an infinite number of particles from the optimal period of the periodic protocol. These optimal thresholds for an infinite number of particles give good results for many particles. In addition, they also give good results for few particles due to the smooth dependence of the current up to these threshold values.

Journal Article↗
PubMed1990

A general method of optimizing automated DNA synthesis to decrease chemical consumption to less than half.

An automated DNA synthesis protocol (FC3) has been optimized to decrease the cost of chemical consumption by two- to threefold. Anion-exchange chromatography at pH 12.5 in a Mono Q (Pharmacia) column was used to analyze the oligonucleotides we synthesized. From these analyses, we formulated the FC3 program and a procedure by which a DNA synthesizer designer or operator can use 18-base-long homopolymers of A, G, C, and T to optimize the reaction times, reagent concentrations, solvent wash conditions, and the many steps in the synthesis cycle.

Automation↗
PubMed1999

Human immunodeficiency virus type 1 envelope-specific cytotoxic T lymphocytes response dynamics after prime-boost vaccine regimens with human immunodeficiency virus type 1 canarypox and pseudovirions.

Virus-specific cytotoxic T lymphocytes (CTLs) may represent significant immune mechanisms in the control of human immunodeficiency virus (HIV) infection and, therefore, CTL induction may be a fundamental goal in the development of an efficacious acquired immunodeficiency syndrome (AIDS) vaccine. In the current study, prime-boost protocols were used to investigate the potential of noninfectious human immunodeficiency virus type 1 (HIV-1) pseudovirions (HIV PSV) in enhancing HIV-specific CTL responses in Balb/c mice primed with the recombinant canarypox vector, vCP205, encoding HIV-1 gp120 (MN strain) in addition to Gag/Protease (HIB strain). The prime-boost immunization regimens were administered intramuscularly and involved injections of vCP205 followed by boosts with HIV PSV. Previous vaccination strategies solely involving vCP205 had induced good cellular immune responses in uninfected human volunteers, despite some limitations. The use of genetically engineered HIV PSV was a logical step in the evaluation of whole noninfectious virus or inactivated virus vaccine strategies, particularly as a potential boosting agent for vCP205-primed recipients. Based on this current study, HIV PSV appeared to have the capability to effectively induce and boost cell-mediated HIV-1-specific responses. In order to observe the immune effects of HIV PSV in a prime-boost immunization strategy, both HIV vaccine immunogens required careful titration in vivo. This suggests that careful consideration should be given to the optimization of immunization protocols destined for human use.

AIDS Vaccines↗
PubMed1999

Three-dimensional computer reconstruction of prostate cancer from radical prostatectomy specimens: evaluation of the model by core biopsy simulation.

OBJECTIVES: A technique was developed for three-dimensional (3D) modeling of prostate cancer and transrectal biopsies. To test the model, the cancer yield of a simulated 10-biopsy protocol was compared with a simulated sextant protocol and with preoperative biopsies regarding cancer detection and correlation with tumor volume. METHODS: Transrectal ultrasound-guided core biopsies were taken from 81 men according to a standardized 10-biopsy protocol that included sextant biopsies. The patients underwent radical prostatectomy and specimens were step-sectioned and whole-mounted. Cancer and the prostate capsule were outlined on the slides and the regions transferred to a computer software program developed by our group. A 3D volume of each prostate was reconstructed from the sections. Virtual core biopsy needles imitating the positions of the real biopsies were inserted into the prostate and the cancer yield was calculated. Only the standardized positions were considered in this study (ie, additional biopsies from hypoechoic foci were not accounted for). RESULTS: Of the cancers detected with 10 standardized virtual biopsies, 24% would have remained undetected with sextant biopsies. The cancer yield of 10 virtual biopsies correlated with the preoperative biopsies (r = 0.64) and with the tumor volume (r = 0.56). A multiple regression analysis showed that the cancer yield of a simulation of 10 biopsies correlated better with tumor volume than did a simulation of sextant biopsies (P = 0.02). CONCLUSIONS: We conclude that computer-assisted 3D reconstruction of prostate cancer can be used as a model for evaluation and optimization of biopsy protocols.

Adult↗
PubMed2005

What is the 'ideal' duration of progesterone supplementation before the transfer of cryopreserved-thawed embryos in estrogen/progesterone replacement protocols?

Different studies dealing with the start of progesterone supplementation in assisted reproduction treatment cycles have shown that the problem apparently is the correct timing. We therefore would like to discuss the data on: (i) the start of progesterone replacement in oocyte donation programmes; (ii) the start of progesterone replacement in frozen-thawed hormone-supplemented cycles; (ii) the problem of too early a rise of progesterone in fresh IVF cycles as a model of too early an administration of progesterone; and (iv) the benefit of high progesterone levels on the day of embryo transfer in fresh IVF cycles. From the data reviewed in this paper it seems to be appropriate to start progesterone administration before transfer in oocyte donation programmes as well as transfer of cryopreserved/thawed cells as soon as the endometrium is developed sufficiently (> or =8 mm, trilaminar pattern), and to perform the embryo transfer not before day 3-4 of progesterone treatment, i.e. embryo development on day 2-3. Studies dealing with the influence of too early a rise of progesterone in fresh IVF cycles have shown different results. In fact high progesterone levels seem to reflect a high response but not a lower probability of conception. Furthermore, high progesterone levels on the day of embryo transfer in fresh IVF cycles could lower myometrial contractility and therefore increase implantation rates. Since the experience from oocyte donation programes shows the benefit of a longer preparation time using progesterone, and high progesterone levels seem to have a benefit during embryo transfer, this would suggest extending progesterone administration before transfer. However, we have to find the optimal individual transfer protocol after mock cycles, for example with pinopode detection or other methods applicable in routine IVF programmes. We need more studies to be sure whether reproductive outcome after transfer of cryopreserved-thawed cells in estrogen/progesterone supplement cycles is influenced by the duration of progesterone pretreatment. If this is so, we must look for practicable methods to modify the protocols according to the individual patient, the embryonic developmental stage during transfer and other variables.

Clinical Trials as Topic↗
PubMed2005

A novel sample labeling method with restriction display PCR for 60-mer oligonucleotide microarray.

OBJECTIVE: To investigate the value of restriction display PCR (RD-PCR) as a novel and expedient sample labeling method for high-density 60-mer oligonucleotide microarray. METHODS: Peripheral blood samples from three volunteers were collected and the total RNA was extracted from the peripheral blood mononuclear cells and labeled with RD-PCR protocol, followed by hybridization with Agilent Human 1B oligonucleotide microarrays in a two-color comparison format. The RNA from the same subject was divided into two aliquot and labeled with Cy3 and Cy5 respectively. The spots with significant difference between the foreground and local background intensities and those without significant difference between Cy5 and Cy3 signal intensities were selected for analysis. SPSS software was used to perform the statistical tests and plot generation. VSN packages were used under R language to remove the systematic array and dye biases. RESULTS: Totally 8744 common spots of the 3 microarrays were evaluated. The results demonstrated that RD-PCR could be a promising novel method for efficient labeling of microarray samples. Further analysis indicated the presence of adjustable biases derived from the array and incorporated dye in the labeling processes. The RD-PCR labeling showed better performance than the conventional approaches in regards to reproducibility of the quantitative signals for gene intensity and capability to label RNAs of lowly expressed genes. CONCLUSION: Given the evidence of the feasibility of using RD-PCR labeling in the field of high-density long oligonucleotide microarray, further optimization of the protocol may unleash the full potential of this novel labeling method.

Humans↗
PubMed1989

[Results of cisplatin-based polychemotherapy and analysis of prognostic factors in ovarian cancer. Apropos of 106 cases].

One hundred and six patients with stage Ic to IV ovarian carcinoma were treated by a protocol consisting of optimal debulking surgery followed by 9 cycles of CHAP chemotherapy. Clinical response was confirmed by a second-look procedure. Sixty-nine patients (65%) responded with 54 histological complete remissions (50.8%). Nineteen patients did not receive any complementary treatment due to a negative reaction, or prolonged neutropenia. Seven patients received maintenance chemotherapy, 10 an abdominal radiotherapy, 22 intraperitoneal chemotherapy and 11 autologous bone marrow transplantation. The 5-year survival rate was 32.5% and disease-free survival rate was 39.7%. Prognostic-factor analysis showed that age, initial staging, residual disease and cytological grading were significant. The authors propose a classification based on the risk of relapse, and different therapeutic indications for improving response rate and patient survival.

Adult↗
PubMed2000

A comparative evaluation of gene transfer into blood cells using the same retroviral backbone for independent expression of the EGFP and deltaLNGFR marker genes.

BACKGROUND AND OBJECTIVES: Retroviral vectors are widely used to deliver foreign genes to hematopoietic stem cells (HSC). Improvement of marking protocols needs reporter genes to allow rapid detection and efficient selection of transduced cells. The great potential of EGFP and LNGFR as reporter systems prompted us to compare them simultaneously, using the same retroviral backbone and the same gene transfer procedures. DESIGN AND METHODS: The EGFP and LNGFR coding sequences were separately cloned into the MFG retroviral backbone. A cloning strategy assuring that both genes utilize the same ATG as the start codon was adopted. Marker gene expression, viral titers, transduction efficiency, and vector stability were evaluated in expanded amphotropic packaging clones and human hematopoietic cell lines by flow cytometry and PCR analysis. Vectors were also tested for their ability to transduce CD34+ peripheral blood cells. RESULTS: A significantly larger number of MFG- LNGFR packaging clones were obtained that produced high viral titers. A direct correlation between viral titer and marker gene expression in packaging clones was demonstrated for both constructs. Similar expression kinetics and absence of in vitro toxicity in transduced cells were also observed for both constructs. Successful infection of CD34+ cells was achieved even after a short time of exposure to recombinant viruses. INTERPRETATION AND CONCLUSIONS: Our results demonstrate that EGFP and LNGFR marker genes are equally useful for a rapid, specific and non-toxic detection of transduced cells. The MFG-EGFP construct appears useful to optimize gene transfer protocols in vitro. On the other hand, the MFG-LNGFR construct, for making possible a more efficient selection of high titer producer clones, as well as for safety and adaptability to the in vivo use, is more suitable for clinical applications.

Cell Line↗
PubMed1996

Photochemical inactivation of duck hepatitis B virus in human platelet concentrates: a model of surrogate human hepatitis B virus infectivity.

BACKGROUND: Photochemical decontamination of platelet concentrates (PCs) has been demonstrated by the use of 8-methoxypsoralen and ultraviolet A light. Systems for studying the inactivation of blood-borne viruses facilitate the evaluation of photochemical decontamination protocols. STUDY DESIGN AND METHODS: Duck hepatitis B virus (HBV), a model for human HBV, was adapted for the study of hepadnavirus inactivation. A highly specific in vitro infectivity assay used primary duck hepatocyte cultures and was followed by the detection of replicated duck HBV sequences. RESULTS: Duck HBV-infected primary duck hepatocyte cultures produced authentic infectious virus. High-titer (> 10(9) virus genome equivalents/mL) duck HBV-infected sera were completely inactivated in serum or PCs by the use of 100 micrograms per mL of 8-methoxypsoralen and 70 J per cm2 of ultraviolet A light. Intracellular duck HBV (> 4.2 log10) in PCs was also inactivated. Culture results were confirmed by a sensitive duckling infectivity assay that indicated that 6.3 log10 of infectious duck HBV had been inactivated by photochemical decontamination. CONCLUSION: The sensitivity of the culture assay was comparable to that of the duckling assay using polymerase chain reaction gene amplification to detect duck HBV. Duck HBV inactivation in PCs was dependent on the dose of ultraviolet A light and independent of 8-methoxypsoralen concentrations of 100 to 300 micrograms per mL: 100 micrograms per mL 8-methoxypsoralen inactivated 4 to 5 log10 of virus in conjunction with 20 to 40 J per cm2 of ultraviolet A light. The polymerase chain reaction-enhanced duck HBV culture system has utility in optimizing photochemical decontamination protocols.

Animals↗
PubMedDate not supplied

Thermal analysis of garlic shoot tips during a vitrification procedure.

The thermal behavior of garlic shoot tips was analyzed during the course of a vitrification protocol using the PVS3 vitrification solution. The size of shoot tips did not significantly influence the thermal behavior of garlic shoot tips. Though there was no significance, endo-thermal enthalpy from melting of crystalline ice increased as preculture duration increased to 6 days. Preculture on medium with 0.5 M sucrose significantly lowered exo- and endothermal enthalpies of dehydration-control shoot tips. By contrast, after dehydration with PVS3 solution, the concentration of sucrose in preculture medium had no significant effect on the value of enthalpies. A big thermal event was observed in garlic shoot tips air-dried for 1-3 h before dehydration. Both vitrification solution and dehydration duration significantly (P < 0.0001) influenced exo- and endothermal enthalpies. After dehydration with PVS1, PVS2, Fahy or Steponkus solutions for 120 min, only a small peak was detected in some shoot tips, but recovery of cryopreserved shoot tips was low. Dehydration duration with PVS3 solution significantly (P < 0.0001) influenced exo- and endothermal enthalpies and onset temperatures during cooling and warming. After dehydration for 150 and 180 min with PVS3 vitrification solution, no crystallization was observed during cooling and warming in most replicates, and recovery of cryopreserved shoot tips was highest (> 80%). There was a significant (P < 0.001) negative correlation between moisture content of shoot tips and concentration of sucrose and glycerol, and regeneration of cryopreserved shoot tips. By contrast, there was a significant (P < 0.001) positive correlation between MC and enthalpy of ice melting, and onset temperature of crystallization. Overall, the results of the analysis of the thermal behavior of garlic shoot tips coincide very well with their recovery after cryopreservation and provide a very useful tool for the establishment and optimization of cryopreservation protocols.

Cryopreservation↗
PubMed2006

Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells.

BACKGROUND: There is a growing interest in cord blood as a source of primitive stem cells with the capacity for multilineage differentiation. Pure cell fractions are needed for the characterization and in vitro expansion of stem cells as well as for their use in preclinical research. However, enrichment of stem cells is challenging due to the lack of stem cell-specific markers and gentle protocols for the isolation of highly pure stem cell fractions. Protocols developed for the enrichment of peripheral blood-derived stem cells have been found to be suboptimal for cord blood. RESULTS: In this study, immunomagnetic cell sorting protocols to purify CD34+, CD133+ and Lin- cells from fresh and cryopreserved cord blood were optimized. Reproducible purities of up to 97% were reached. The selected cells were highly viable having substantial colony-forming potential. CONCLUSION: The optimized protocols enable rapid enrichment of highly pure hematopoietic stem cells from both fresh and cryopreserved cord blood.

AC133 Antigen↗
PubMed2026

Diagnostic performance of panfungal PCR on tissue specimens for the diagnosis of invasive fungal diseases: a systematic review and meta-analysis of the Fungal PCR Initiative (FPCRI).

UNLABELLED: Invasive fungal diseases are difficult to diagnose because of the limited sensitivity of culture. Panfungal PCR amplicon sequencing assays (targeting ribosomal RNA, such as 18S, 28S, ITS) are recommended for fungal identification in histopathology samples showing fungal elements. However, data describing its overall performance and consistency are lacking. This systematic literature review and meta-analysis assessed the performance of panfungal PCR on formalin-fixed paraffin-embedded (FFPE) and non-fixed (fresh or frozen) tissue samples. A systematic literature search was performed to include studies reporting the use of panfungal PCR for fungal identification in FFPE or non-fixed tissue samples. PCR sensitivity and specificity were assessed using the reference standard of histopathology showing fungal elements. Quality assessment was performed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Pooled estimates were obtained using random-effects meta-analysis. Twenty-eight studies were included. In FFPE samples (18 studies, 852 samples), sensitivity and specificity were 75.4% (95% confidence interval [CI], 59.2-86.6) and 93.5% (70.2-98.9), respectively. Sensitivity in non-fixed samples (13 studies, 207 samples) was 86.5% (74.7-93.3), while specificity could not be assessed (insufficient data). Comparative analyses showed a significantly higher sensitivity of panfungal PCR over culture (88.2%; 76-94.7 vs 52.2%; 39-65, P = 0.001). Sub-analyses could not demonstrate the superiority of one PCR target over another due to limited data. Panfungal PCR exhibited adequate sensitivity and good specificity in FFPE samples. Sensitivity was even higher in non-fixed samples and largely superior to culture. Nevertheless, large interstudy variability was observed, warranting interlaboratory studies to define the optimal PCR target and standardized protocols. IMPORTANCE: Invasive fungal diseases are difficult to diagnose because of the low sensitivity of culture. Panfungal PCRs are widely used for fungal identification in tissue specimens but suffer from heterogeneous procedures and performance. This meta-analysis shows an acceptable sensitivity (75.4% and 86.5% in fixed and non-fixed samples, respectively) and good specificity (93.5%) of panfungal PCR, supporting its use, not only on histopathology-positive fixed samples but also in non-fixed samples concomitantly with other diagnostic tools (cultures and fungal-specific PCRs if available). These results provide a strong basis for further standardization of panfungal PCR techniques via interlaboratory assays to assess reproducibility and optimize analytical protocols. CLINICAL TRIALS: This study is registered with PROSPERO as CRD42023461148.

Humans↗
PubMed1981

The economics of cancer prevention and detection: getting more for less.

Economics has been described as the science of allocating scarce resources. Given the magnitude of the cancer problem and the obvious impracticality of eliminating every carcinogen or screening every person for every disease, our success in preventing cancer will depend greatly on our ability to use available resources efficiently. This paper will illustrate the principles of economics applied to the early detection of cancer of the lung, breast, colon, and cervix. The objective is to deliver the highest benefit possible. The available options include choice of the population to be screened (e.g., ages and risk factors), the tests to be used, and the frequency of examinations. The issues to be considered in making these choices include the expected benefits of early detection, the risks and side effects of the tests, the acceptibility of the program, the costs of the tests, the consequences of false positives, changes in therapy, and decrease in disability and lost earnings. The final choice of an "optimal" early detection protocol requires estimating and comparing these and other outcomes. This is a question of expectations and values; there is no "correct" answer or "best" program. These issues also have implications for the feasibility and prospects for third-party reimbursement for cancer prevention activities.

Adult↗