Intellicount: High-Throughput Quantification of Fluorescent Synaptic Protein Puncta by Machine Learning.

Synapse formation analyses can be performed by imaging and quantifying fluorescent signals of synaptic markers. Traditionally, these analyses are done using simple or multiple thresholding and segmentation approaches or by labor-intensive manual analysis by a human observer. Here, we describe Intellicount, a high-throughput, fully-automated synapse quantification program which applies a novel machine learning (ML)-based image processing algorithm to systematically improve region of interest (ROI) identification over simple thresholding techniques. Through processing large datasets from both human and mouse neurons, we demonstrate that this approach allows image processing to proceed independently of carefully set thresholds, thus reducing the need for human intervention. As a result, this method can efficiently and accurately process large image datasets with minimal interaction by the experimenter, making it less prone to bias and less liable to human error. Furthermore, Intellicount is integrated into an intuitive graphical user interface (GUI) that provides a set of valuable features, including automated and multifunctional figure generation, routine statistical analyses, and the ability to run full datasets through nested folders, greatly expediting the data analysis process.

Non-invasive risk assessment of fetal sex chromosome aneuploidy through directed analysis and incorporation of fetal fraction.

OBJECTIVE: To assess the performance of a directed chromosomal analysis approach in the prenatal evaluation of fetal sex chromosome aneuploidy. METHODS: We analyzed 432 frozen maternal plasma samples obtained from patients prior to undergoing fetal diagnostic testing. The cohort included women greater than 18 years of age with a singleton pregnancy of greater than 10 weeks gestation. Samples were analyzed using a chromosome-selective approach (DANSR(TM) ) and a risk algorithm that incorporates fetal fraction (FORTE(TM) ). RESULTS: The cohort included 34 cases of sex chromosome aneuploidy. The assay correctly identified 26 of 27 (92.6%) cases of Monosomy X, one case of XXX, and all six cases of XXY. There were four false positive cases of sex chromosome aneuploidy among 380 euploid cases for an overall false positive rate of less than 1%. DISCUSSION: Analysis of the risk for sex chromosome aneuploidies can be accomplished with a targeted assay with high sensitivity.

The impact of supplemental dietary methionine sources on volatile compound concentrations in broiler excreta.

The impact of different Met sources on broiler fecal odor volatiles was determined by evaluating the types of sulfur compounds produced in broiler excreta. Two experiments were conducted using straight-run broiler chicks randomly distributed in battery cages, with 3 replicate pens of 16 birds each. The treatment groups were 1) dry Met hydroxy analogue (dry MetHA), 2) sodium methioninate aqueous solution (NaMet), 3) liquid Met hydroxy analogue (Liq MetHA), 4) D,L- Met, and 5) no supplemental Met (control group). The Met activities of each Met source were 52, 45.9, 88, and 98%, respectively. All diets were formulated to contain either 0.8% (experiment 1) total Met activity or 0.5% Met activity in the starter and 0.38% Met activity in the grower (experiment 2) (except the control group, 0.35% Met activity), but otherwise met NRC nutrient requirements (NRC, 1994). Diets were fed ad libitum from d 1 to 6 wk of age. There were no significant differences in BW among the treatments. All excreta were collected in litter pans lined with aluminum foil. In experiment 1, at wk 6, broiler excreta were collected for a 24-h period, and 4.5 g of broiler excreta from each treatment group was collected into 15-mL headspace vials. Samples were analyzed by gas chromatography/mass spectrometry (GC/MS). The volatile sulfur compounds that were identified and quantified in the broiler excreta were H2S, carbonyl sulfide (COS), methyl mercaptan (CH3SH), dimethyl disulfide (CH3SSCH3), and dimethyl trisulfide (CH3SSSCH3). The NaMet treatment group had significantly higher concentrations of H2S, COS, and CH3SSCH3 compared with all other treatment groups. The Liq MetHA group had significantly lower concentrations of H2S, COS, CH3SH, and CH3SSCH3 compared with the other treatment groups. The dry MetHA group significantly had the highest concentration of CH4SH. The D,L-Met treatment group had the significantly highest concentration of CH3SSSCH3 and the lowest concentration of H2S. The control group had the significantly lowest concentrations of CH3SH, CH3SSCH3, and CH3SSSCH3 compared with the other treatment groups. In experiment 2, at wk 6, an electronic nose was used to evaluate 15 air samples per treatment group. In addition, 15 air samples (containing 6 to 8 L of air in a Tedlar bag, 3 samples per treatment group) were collected for odor evaluation by a sensory panel. Electronic nose sensor data revealed that volatile compounds in broiler excreta from the control group were significantly different from the other 4 treatment groups. Evaluation of the air samples by a sensory panel determined that there was a statistically significant difference in odor threshold detection between the control group and the other treatment groups. The dilutions to threshold of control group, NaMet, dry MetHA, Liq MetHA, and D,L-Met were 350, 492, 568, 496, and 526 odor units, respectively. These findings demonstrate that dietary Met sources significantly influenced odorous volatile concentrations in broiler excreta.

Membrane-associated quinoprotein formaldehyde dehydrogenase from Methylococcus capsulatus Bath.

A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 micromol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)(+)-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159-167, 1999; Vorholt et al., J. Bacteriol. 180:5351-5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b(559/569) complex as the physiological electron acceptor.

Functional classification of swine manure management systems based on effluent and gas emission characteristics.

Gaseous emissions from swine (Sus scrofa) manure storage systems represent a concern to air quality due to the potential effects of hydrogen sulfide, ammonia, methane, and volatile organic compounds on environmental quality and human health. The lack of knowledge concerning functional aspects of swine manure management systems has been a major obstacle in the development and optimization of emission abatement technologies for these point sources. In this study, a classification system based on gas emission characteristics and effluent concentrations of total phosphorus (P) and total sulfur (S) was devised and tested on 29 swine manure management systems in Iowa, Oklahoma, and North Carolina in an effort to elucidate functional characteristics of these systems. Four swine manure management system classes were identified that differed in effluent concentrations of P and S, methane (CH4) emission rate, odor intensity, and air concentration of volatile organic compounds (VOCs). Odor intensity and the concentration of VOCs in air emitted from swine manure management systems were strongly correlated (r2 = 0.88). The concentration of VOC in air samples was highest with outdoor swine manure management systems that received a high input of volatile solids (Type 2). These systems were also shown to have the highest odor intensity levels. The emission rate for VOCs and the odor intensity associated with swine manure management systems were inversely correlated with CH4 and ammonia (NH3) emission rates. The emission rates of CH4, NH3, and VOCs were found to be dependent upon manure loading rate and were indirectly influenced by animal numbers.

Correlation of human olfactory responses to airborne concentrations of malodorous volatile organic compounds emitted from swine effluent.

Direct multicomponent analysis of malodorous volatile organic compounds (VOCs) present in ambient air samples from 29 swine (Sus scrofa) production facilities was used to develop a 19-component artificial swine odor solution that simulated olfactory properties of swine effluent. Analyses employing either a human panel consisting of 14 subjects or gas chromatography were performed on the air stream from an emission chamber to assess human olfactory responses or odorant concentration, respectively. Analysis of the olfactory responses using Fisher's LSD statistics showed that the subjects were sensitive to changes in air concentration of the VOC standard across dilutions differing by approximately 16%. The effect of chemical synergisms and antagonisms on human olfactory response magnitudes was assessed by altering the individual concentration of nine compounds in artificial swine odor over a twofold concentration range while maintaining the other 18 components at a constant concentration. A synergistic olfactory response was observed when the air concentration of acetic acid was increased relative to the concentration of other VOC odorants in the standard. An antagonistic olfactory response was observed when the air concentration of 4-ethyl phenol was increased relative to the other VOC odorants in the standard. The collective odorant responses for nine major VOCs associated with swine odor were used to develop an olfactory prediction model to estimate human odor response magnitudes to swine manure odorants through measured air concentrations of indicator VOCs. The results of this study show that direct multicomponent analysis of VOCs emitted from swine effluent can be applied toward estimating perceived odor intensity.

Abatement of ammonia and hydrogen sulfide emissions from a swine lagoon using a polymer biocover.

The purpose of this research was to determine the efficiency of a polymer biocover for the abatement of H2S and NH3 emissions from an east-central Missouri swine lagoon with a total surface area of 7800 m2. The flux rate of NH3, H2S, and CH4 was monitored continuously from two adjacent, circular (d = 66 m) control and treatment plots using a nonintrusive, micrometeorological method during three independent sampling periods that ranged between 52 and 149 hr. Abatement rates were observed to undergo a temporal acclimation event in which NH3 abatement efficiency improved from 17 to 54% (p = < 0.0001 to 0.0005) and H2S abatement efficiency improved from 23 to 58% (p < 0.0001) over a 3-month period. The increase in abatement efficiency for NH3 and H2S over the sampling period was correlated with the development of a stable anaerobic floc layer on the bottom surface of the biocover that reduced mass transfer of NH3 and H2S across the surface. Analysis of methanogenesis activity showed that the biocover enhanced the rate of anaerobic digestion by 25% when compared with the control. The biocover-enhanced anaerobic digestion process was shown to represent an effective mechanism to counteract the accumulation of methanogenic substrates in the biocovered lagoon.

Microchannel DNA sequencing matrices with a thermally controlled "viscosity switch".

Polymers and hydrogels that swell or shrink in response to environmental stimuli such as changes in temperature, pH, or ionic strength are of interest as switchable materials for applications in biotechnology. In this paper, we show that thermoresponsive polymers offer some particular advantages as entangled matrices for DNA sequencing by capillary and microchip electrophoresis. Matrices based on conventional water-soluble polymers demand a compromise in their design for microchannel electrophoresis: whereas highly entangled solutions of high molar mass polymers provide optimal sequencing performance, their highly viscous solutions require application of high pressures to be loaded into electrophoresis microchannels. Here, we demonstrate the reproducible synthesis, precise characterization, and excellent DNA sequencing performance of high molar mass, thermoresponsive polymer matrices that exhibit a reversible, temperature-controlled "viscosity switch" from high-viscosity solutions at 25 degrees C to low-viscosity, microphase-separated colloidal dispersions at a chosen, elevated temperature. The viscosity switch decouples matrix loading and sieving properties, enabling acceleration of microchannel flow by 3 orders of magnitude. DNA sequencing separations yielding read lengths of 463 bases of contiguous sequence in 78 min with 97% base-calling accuracy can be achieved in these matrices. Switchable matrices will be particularly applicable to microfluidic devices with dynamic temperature control, which are likely to provide the next major leap in the efficiency of high-throughput DNA analysis.

Rapid method to estimate the presence of secondary metabolites in microbial extracts.

Screening microbial secondary metabolites is an established method to identify novel biologically active molecules. Preparation of biological screening samples from microbial fermentation extracts requires growth conditions that promote synthesis of secondary metabolites and extraction procedures that capture the secondary metabolites produced. High-performance liquid chromatography (HPLC) analysis of fermentation extracts can be used to estimate the number of secondary metabolites produced by microorganisms under various growth conditions but is slow. In this study we report on a rapid (approximately 1 min per assay) surrogate measure of secondary metabolite production based on a metabolite productivity index computed from the electrospray mass spectra of samples injected directly into a spectrometer. This surrogate measure of productivity was shown to correlate with an HPLC measure of productivity with a coefficient of 0.78 for a test set of extracts from 43 actinomycetes. This rapid measure of secondary metabolite productivity may be used to identify improved cultivation and extraction conditions by analyzing and ranking large sets of extracts. The same methods may also be used to survey large collections of extracts to identify subsets of highly productive organisms for biological screening or additional study.

Use of direct-infusion electrospray mass spectrometry to guide empirical development of improved conditions for expression of secondary metabolites from actinomycetes.

A major barrier in the discovery of new secondary metabolites from microorganisms is the difficulty of distinguishing the minor fraction of productive cultures from the majority of unproductive cultures and growth conditions. In this study, a rapid, direct-infusion electrospray mass spectrometry (ES-MS) technique was used to identify chemical differences that occurred in the expression of secondary metabolites by 44 actinomycetes cultivated under six different fermentation conditions. Samples from actinomycete fermentations were prepared by solid-phase extraction, analyzed by ES-MS, and ranked according to a chemical productivity index based on the total number and relative intensity of ions present in each sample. The actinomycete cultures were tested for chemical productivity following treatments that included nutritional manipulations, autoregulator additions, and different agitation speeds and incubation temperatures. Evaluation of the ES-MS data from submerged and solid-state fermentations by paired t test analyses showed that solid-state growth significantly altered the chemical profiles of extracts from 75% of the actinomycetes evaluated. Parallel analysis of the same extracts by high-performance liquid chromatography-ES-MS-evaporative light scattering showed that the chemical differences detected by the ES-MS method were associated with growth condition-dependent changes in the yield of secondary metabolites. Our results indicate that the high-throughput ES-MS method is useful for identification of fermentation conditions that enhance expression of secondary metabolites from actinomycetes.

Identification of intermediates of in vivo trichloroethylene oxidation by the membrane-associated methane monooxygenase.

The rate and products of trichloroethylene (TCE) oxidation by Methylomicrobium album BG8 expressing membrane-associated methane monooxygenase (pMMO) were determined using 14C radiotracer techniques. [(14)C]TCE was degraded at a rate of 1.24 nmol (min mg protein)(-1) with the initial production of glyoxylate and then formate. Radiolabeled CO(2) was also found after incubating M. album BG8 for 5 h with [(14)C]TCE. Experiments with purified pMMO from Methylococcus capsulatus Bath showed that TCE could be mineralized to CO(2) by pMMO. Oxygen uptake studies verified that M. album BG8 could oxidize glyoxylate and that pMMO was responsible for the oxidation based on acetylene inactivation studies. Here we propose a pathway of TCE oxidation by pMMO-expressing cells in which TCE is first converted to TCE-epoxide. The epoxide then spontaneously undergoes HCl elimination to form glyoxylate which can be further oxidized by pMMO to formate and CO(2).

Primary structure of cytochrome c' of Methylococcus capsulatus Bath: evidence of a phylogenetic link between P460 and c'-type cytochromes.

Cytochrome c' of Methylococcus capsulatus Bath is involved in electron flow from the enzyme responsible for hydroxylamine oxidation, cytochrome P460, to cytochrome C555. This cytochrome is spectrally similar to other cytochromes c' but is larger (16,000 Da) and has a lower midpoint potential (-205 mV). By a combination of Edman degradation, mass spectroscopy, and gene sequencing, we have obtained the primary structure of cytochrome c' from M. capsulatus Bath. The cytochrome shows low sequence similarity to other cytochromes c', only residues R12, Y53, G56, and the C-terminal heme-binding region (GXXCXXCHXXXK) being conserved. In contrast, cytochrome c' from M. capsulatus Bath shows considerable sequence similarity to cytochromes P460 from M. capsulatus Bath (31% identity) and from Nitrosomonas europaea (18% identity). This suggests that P460-type cytochromes may have originated from a c'-type cytochrome which developed a covalent cross-link between a lysine residue and the c'-heme.

Comparison of non-invasive blood pressure measurements on the arm and calf during cesarean delivery.

OBJECTIVE: Shivering may occur in 75% of women undergoing spinal anesthesia for cesarean delivery and may render an automated noninvasive blood pressure (ANIBP) device incapable of determining blood pressurc (BP). When patients shiver under spinal anesthesia, the lower extremities do not exhibit the same involuntary muscle movements as do the upper extremities. This study was undertaken to determine if a correlation exists between ANIBP measurements in the arm and calf of women undergoing cesarean delivery under spinal anesthesia. METHODS: We enrolled 73 women in this blinded, prospective study. Simultaneous arm and calf BP were measured with an ANIBP and differences between the two were determined. RESULTS: We found significant differences between the average difference in systolic and in diastolic BP, no significant difference between the average mean BP, and a tendency for the systolic BP to be higher and the diastolic BP to be lower in the calf than in the arm; however, there was a large degree of variability among patients. CONCLUSION: We conclude that there is a poor correlation between the BP measured by an ANIBP on the calf and one on the arm. In the parturient undergoing cesarean section, lower extremity BP as measured by an ANIBP does not correlate with the arm ANIBP and should not be used to assure fetal well being.

Improved epidural analgesia in the parturient in the 30 degree tilt position.

PURPOSE: To compare the incidence of incomplete analgesia when epidural local anesthetic is administered with the parturient supine in a 30 degree leftward tilt or in the left lateral decubitus position. METHODS: After placement of a multiorifice catheter 5 cm into the epidural space, 293 women in active labour were randomly positioned either to the left lateral decubitus position (lateral group) or supine with a 30 degree leftward tilt (tilt group) and then received 13 mL bupivacaine 0.25%. The success of the epidural block was determined by asking the patient if she required additional medication 15 min later. The incidence of complications (fetal heart rate decelerations, hypotension, and ephedrine usage) was noted. RESULTS: In the lateral group, 38% required additional medication compared with 24% in the tilt group (P = 0.006). There were no differences between groups in the incidence of maternal hypotension or fetal heart rate decelerations, but more women (10%) received ephedrine in the lateral than in the tilt group (4%), P = 0.035. CONCLUSIONS: Placing the parturient supine with a 30 degree leftward tilt is associated with a greater success rate of labour epidural analgesia without an increase in complications than in women in the left lateral decubitus position. This advantage should be considered when positioning the parturient after epidural catheter placement.

Epidural ropivacaine for the initiation of labor epidural analgesia: a dose finding study.

UNLABELLED: The purpose of our study was to determine the lowest concentration of ropivacaine that offers pain relief for the initiation of labor epidural analgesia. Women in active labor were enrolled in this prospective, randomized, double-blinded study to receive either ropivacaine 0.20% (Group I), ropivacaine 0.15% (Group II), or ropivacaine 0.10% (Group III). After placement of the epidural catheter, 13 mL of the study medication was administered. Fifteen minutes later, the adequacy of analgesia was assessed. If the woman reported that her degree of analgesia was not adequate, an additional 5 mL of the study medication was given, the degree of pain relief was reassessed 15 min later, and the study was concluded. A sequential study design was used to assess the success rates. We found that 26 of 28 (93%) women in Group I had adequate analgesia, compared with only 18 of 28 (64%) in Group II (P = 0.014) and 4 of 12 (33%) in Group III (P = 0.003). We conclude that ropivacaine 0.20% offers adequate analgesia significantly more often than either ropivacaine 0.15% or ropivacaine 0.10%. If one selects ropivacaine as the sole local anesthetic for the initiation of labor epidural analgesia, the minimal concentration should be 0.20%. IMPLICATIONS: The lowest effective concentration of ropivacaine for the initiation of labor epidural analgesia has not been determined. We found that ropivacaine 0.20% offers adequate analgesia significantly more often than either ropivacaine 0.15% or ropivacaine 0.10%. If one selects ropivacaine as the sole local anesthetic for the initiation of labor epidural analgesia, the minimal concentration should be 0.20%.

High-molecular-mass multi-c-heme cytochromes from Methylococcus capsulatus bath.

The polypeptide and structural gene for a high-molecular-mass c-type cytochrome, cytochrome c553O, was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6. 0. The heme c concentration was estimated to be 8.2 +/- 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c553O was used to identify a DNA fragment from M. capsulatus Bath that contains occ, the gene encoding cytochrome c553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118, 620 Da that shows approximately 40% amino acid sequence identity with occ and contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94, 000 Da and contains seven c-heme-binding motifs but shows no sequence homology to occ or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome.

Cytochrome P460 genes from the methanotroph Methylococcus capsulatus bath.

P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium Nitrosomonas europaea (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231-244, 1972) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879-5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from M. capsulatus Bath that contains cyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12, 000-Da periplasmic protein, and the third (cyp) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal, c-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457-460, 1997). Gene probing using cyp indicated that a cytochrome P460 similar to that from M. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP but not in the type I methanotrophs Methylobacter marinus A45, Methylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium.

Copper-binding compounds from Methylosinus trichosporium OB3b.

Two copper-binding compounds/cofactors (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMOC) mutant, PP319 (P. A. Phelps et al., Appl. Environ. Microbiol. 58:3701-3708, 1992), of Methylosinus trichosporium OB3b. Both CBCs are small polypeptides with molecular masses of 1,218 and 779 Da for CBC-L1 and CBC-L2, respectively. The amino acid sequence of CBC-L1 is S?MYPGS?M, and that of CBC-L2 is SPMP?S. Copper-free CBCs showed absorption maxima at 204, 275, 333, and 356 with shoulders at 222 and 400 nm. Copper-containing CBCs showed a broad absorption maximum at 245 nm. The low-temperature electron paramagnetic resonance (EPR) spectra of copper-containing CBC-L1 showed the presence of a copper center with an EPR splitting constant between those of type 1 and type 2 copper centers (g = 2.087, g = 2.42 G, A = 128 G). The EPR spectrum of CBC-L2 was more complex and showed two spectrally distinct copper centers. One signal can be attributed to a type 2 Cu2+ center (g = 2.073, g = 2.324 G, A = 144 G) which could be saturated at higher powers, while the second shows a broad, nearly isotropic signal near g = 2.063. In wild-type strains, the concentrations of CBCs in the spent media were highest in cells expressing the pMMO and stressed for copper. In contrast to wild-type strains, high concentrations of CBCs were observed in the extracellular fraction of the sMMOC mutants PP319 and PP359 regardless of the copper concentration in the culture medium.

Treatment of incomplete analgesia after placement of an epidural catheter and administration of local anesthetic for women in labor.

BACKGROUND: Approximately 15% of women still have pain after placement of an epidural catheter and administration of local anesthetic for labor analgesia. Two techniques frequently used to treat this pain were compared: (1) withdrawal of the catheter 1 cm and repeated dosing with additional local anesthetic, and (2) repeated dosing with additional local anesthetic without any catheter manipulation. METHODS: Fifteen minutes after placement of a multiple-orifice epidural catheter 5 cm into the epidural space and administration of 13 ml 0.25% bupivacaine to the parturient in labor, the adequacy of analgesia was assessed. All women who had incomplete analgesia were randomized (first intervention) to receive an additional 5 ml 0.25% bupivacaine (local-anestheticonly group) or to receive 5 ml 0.25% bupivacaine after first withdrawing the epidural catheter 1 cm (catheter-manipulation group). If after 15 min the woman still had pain, then (second intervention) the catheter was withdrawn 1 cm and an additional 5 ml 0.25% bupivacaine was administered to the local-anesthetic-only group, whereas 5 ml 0.25% bupivacaine was given to the catheter-manipulation group without further catheter manipulation. The success rate of the second intervention was assessed 15 min later. RESULTS: Seventy-eight women were enrolled in the study, 39 to each group. In the local-anesthetic-only group, 29 (74%) women were successfully treated with the first intervention and the remaining 10 (100%) were successfully treated with the second intervention. In the catheter-manipulation group, 30 (77%) were successfully treated with the first intervention and 7 (100%; 2 patients were not studied because of investigator error) were successfully treated with the second intervention (P=NS). CONCLUSIONS: Administration of additional local anesthetic without first withdrawing the epidural catheter will effectively treat most women for whom analgesia is incomplete after the placement of an epidural catheter during labor.