Defining competence in obstetric epidural anaesthesia for inexperienced trainees.

BACKGROUND: Cumulative sum (CUSUM) analysis has been used for assessing competence of trainees learning new technical skills. One of its disadvantages is the required definition of acceptable and unacceptable success rates. We therefore monitored the development of competence amongst trainees new to obstetric epidural anaesthesia in a large public hospital. METHODS: Obstetric epidural data were collected prospectively between January 1996 and December 2011. Success rates for inexperienced trainees were calculated retrospectively for (1) the whole database, (2) for each consecutive attempt and (3) each trainee's individual overall success rate. Acceptable and unacceptable success rates were defined and CUSUM graphs generated for each trainee. Competence was assessed for each trainee and the number of attempts to reach competence recorded. RESULTS: Mean (sd) success rate for all inexperienced trainees was 76.8 (0.1%), range 63-90%. Consecutive attempt success rate produced a learning curve with a mean success rate commencing at 58% on attempt 1. After attempt 10 the attempt number had no effect on subsequent success rates. From these results, the acceptable and unacceptable success rates were set at 65 and 55% respectively. CUSUM graphs demonstrated 76 out of 81 trainees competent after a mean of 46 (22) attempts. CONCLUSIONS: CUSUM is useful for assessing trainee epidural competence. Trainees require approximately 50 attempts, as defined by CUSUM, to reach competence.

Effectiveness of etoposide chemomobilization in lymphoma patients undergoing auto-SCT.

The effectiveness of stem cell mobilization with G-CSF in lymphoma patients is suboptimal. We reviewed our institutional experience using chemomobilization with etoposide (VP-16; 375 mg/m(2) on days +1 and +2) and G-CSF (5 µg/kg twice daily from day +3 through the final day of collection) in 159 patients with lymphoma. This approach resulted in successful mobilization (>2 × 10(6) CD34+ cells collected) in 94% of patients (83% within 4 apheresis sessions). Fifty-seven percent of patients yielded at least 5 × 10(6) cells in 2 days and were defined as good mobilizers. The regimen was safe with a low rate of rehospitalization. Average costs were $14 923 for good mobilizers and $27 044 for poor mobilizers (P<0.05). Using our data, we performed a 'break-even' analysis that demonstrated that adding two doses of Plerixafor to predicted poor mobilizers at the time of first CD34+ cell count would achieve cost neutrality if the frequency of good mobilizers were to increase by 21%, while the frequency of good mobilizers would need to increase by 25% if three doses of Plerixafor were used. We conclude that chemomobilization with etoposide and G-CSF in patients with lymphoma is effective, with future opportunities for cost-neutral improvement using novel agents.

Minimising the risk of accidental dural puncture with epidural analgesia for labour: a retrospective review of risk factors.

BACKGROUND: Accidental dural puncture has a quoted incidence of between 0.19% and 3.6% of obstetric epidurals and is associated with significant morbidity. We set out to determine possible factors associated with an increased risk of accidental dural puncture. METHODS: We performed a retrospective review of 18385 epidurals, performed over a 15-year period. Factors analysed were: time of day of insertion, loss-of-resistance technique, maternal position, cervical dilatation, grade of anaesthetist and depth to the epidural space. RESULTS: Using univariate analyses we found no association between the risk of accidental dural puncture and the following variables: time of day of insertion (P=0.71), loss-of-resistance technique (P=0.22), maternal position for insertion (P=0.83), degree of cervical dilatation (P=0.41) and grade of anaesthetist performing the epidural (P=0.34). Conversely, we found that the risk of accidental dural puncture increased with increasing depth to the epidural space. This was confirmed using a logistic regression analysis, from which it was estimated that, for every 1-cm increase in depth, the risk of accidental dural puncture increased by approximately 19% (P=0.019; 95% CI for OR: 1.029-1.38). CONCLUSION: We conclude that the risk of accidental dural puncture increases with increasing depth to the epidural space. We suggest further study is required to correlate this risk with increasing body mass index.

L-selectin is dispensable for T regulatory cell function postallogeneic bone marrow transplantation.

In murine models, the adoptive transfer of CD4(+) /CD25(+) regulatory T cells (T(regs) ) inhibited graft-versus-host disease (GvHD). Previous work has indicated a critical role for the adhesion molecule L-selectin (CD62L) in the function of T(regs) in preventing GvHD. Here we examined the capacity of naive wild-type (WT), CD62L(-/-) and ex vivo expanded CD62L(Lo) T(regs) to inhibit acute GvHD. Surprisingly, we found that CD62L(-/-) T(regs) were potent suppressors of GvHD, whereas CD62L(Lo) T(regs) were unable to inhibit disease despite being functionally competent to suppress allo T cell responses in vitro. Concomitant with improved outcomes, WT and CD62L(-/-) T(regs) significantly reduced liver pathology and systemic pro-inflammatory cytokine production, although CD62L(-/-) T(regs) were less effective in reducing lung pathology. While accumulation of CD62L(-/-) T(regs) in GvHD target organs was equivalent to WT T(regs) , CD62L(-/-) T(regs) did not migrate as well as WT T(regs) to peripheral lymph nodes (PLNs) over the first 2 weeks posttransplantation. This work demonstrated that CD62L was dispensable for T(reg) -mediated protection from GvHD.

Serum lactate and base deficit as predictors of mortality after ruptured abdominal aortic aneurysm repair.

OBJECTIVE: Whole body hypoperfusion and lower torso ischaemia-reperfusion contribute to post-operative organ dysfunction in patients undergoing repair of ruptured abdominal aortic aneurysm (AAA). Serum lactate and base deficit are markers of tissue ischaemia and are used to assess the adequacy of resuscitation. This study examines the prognostic value of immediate post-operative levels of serum lactate and base deficit in ruptured AAA. METHODS: Thirty patients (24 men and 6 women of median age 74, range 51-85, years) who survived to at least 12h after ruptured AAA repair were studied retrospectively. The relationship between immediate post-operative lactate, base deficit and mortality was determined. RESULTS: Fifteen patients (50%) died, all from organ failure. An elevated lactate (>2.1 mmol/l) and base deficit (<-2 mmol/l) were present in 20 (67%) and 27 (90%) patients, respectively. Lactate (p<0.001) and base deficit (p=0.003) were significantly higher in non-survivors compared with survivors. Lactate (p=0.021) and base deficit levels (p=0.028) were independently significant for predicting mortality and a significant interaction existed between lactate and base deficit levels for predicting mortality (p=0.027). The sensitivity and specificity of lactate > or =4.0 mmol/l was 13 of 15 (87%) and 12 of 15 (80%), respectively, and base deficit < or =-7 mmol/l was 12 of 15 (80%) and 12 of 15 (80%), respectively. The likelihood ratios for a positive result with the defined cut-off values for lactate and base deficit were 4.3 and 4.0, respectively. Lactate > or =4.0 mmol/l and base deficit or =-7 mmol/l were associated with a 4% probability of death. CONCLUSION: These data demonstrate that an immediate post-operative serum lactate > or =4.0 mmol/l and base deficit < or =-7 mmol/l are good predictors of outcome after ruptured AAA repair. The prognostic value of these simple and inexpensive tests require corroboration in a larger prospective study.

Amplification and detection of transposon insertion flanking sequences using fluorescent muAFLP.

The amplification of transposon insertionflanking sequences is the basis of a variety of techniques usedfor the detection and characterization of specific transposon insertion events. We have developed a method for the efficient size determination and quantification of amplified genomic sequences thatflank Mutator (Mu) transposon insertions in maize. Using this detection method, we have been able to optimize Mu insertion site amplification and to assess amplification from increasingly complex templates representing increasing numbers of Mu-active maize plants. This detection method should be applicablefor the characterization of transposon or transgene insertion events in a wide variety of organisms.

Correlating obstetric epidural anaesthesia performance and psychomotor aptitude.

We investigated the correlation between the scores attained on computerised psychometric tests, measuring psychomotor and information processing aptitudes, and learning obstetric epidural anaesthesia. Ten anaesthetic trainees performed an adaptive tracking task (ADTRACK 3) and one information management task (MAZE) from the MICROPAT testing system. They then embarked on a standardised obstetric anaesthesia training programme prior to performing obstetric on-call duties. The success or failure of their first 50 obstetric epidurals was recorded. There was a significant correlation between mean obstetric epidural failure rate for the second 25 consecutive epidurals and ADTRACK 3 (r = -0.579, p = 0.04) scores. The correlation between the means of the first 25 and 50 consecutive epidurals and ADTRACK 3 scores was not significant. There was no significant correlation between epidural failure rate and MAZE scores. The ratios of the mean epidural failure rate for the last 25 epidurals to the mean for the first 25 epidurals were not significantly correlated with ADTRACK 3 or MAZE scores. Psychomotor abilities appear to be poor determinants of trainees' initial proficiency at obstetric epidural anaesthesia or of trainees' rates of progress during early obstetric epidural training, but may be determinants of an individual's performance after the initial training phase.

Interaction of non-myristoylated NADH-cytochrome b5 reductase with cytochrome b5-dimyristoylphosphatidylcholine vesicles.

An expression vector for NADH-cytochrome b5 reductase containing a thrombin cleavage site directly before the N-terminal glycine residue of the flavoprotein was used to isolate the non-myristoylated enzyme by thrombin cleavage of the initial fusion protein of a short segment of the multiple cloning site of the plasmid vector and the reductase. This flavoprotein preparation, containing only the 28-residue N-terminal peptide segment of the membrane-binding domain of the mammalian enzyme, binds to phospholipid vesicles and interacts with membrane-bound cytochrome b5. The effect of N-myristoylation of the enzyme therefore appears to be limited to facilitating and stabilizing interactions with phospholipid vesicles. However, the relatively short intervening peptide sequence that separates the crucial peptide membrane-binding domain from lysine 41, which has been implicated in the active-site interaction with cytochrome b5 (Strittmatter, P., Kittler, J. M., Coghill, J. E., and Ozols, J. (1992) J. Biol. Chem. 267, 2519-2523), provides some limitation of the distance from the membrane surface for the interactions required for rapid electron transfer from the flavin of the reductase to the heme of cytochrome b5.

Characterization of the role of lysine 110 of NADH-cytochrome b5 reductase in the binding and oxidation of NADH by site-directed mutagenesis.

An expression vector for bovine NADH-cytochrome b5 reductase was used for site-directed mutagenesis of lysine 110, the residue previously implicated in NADH interactions with this flavoprotein. Replacement of this basic residue with an uncharged glutamine resulted in an increase of 3 orders of magnitude in the Km for NADH and a decrease in kcat of an order of magnitude, strongly implicating lysine 110 in both binding of NADH to the reductase and the orientation of the reduced nicotinamide group for rapid hydride ion transfer to the flavin. Substitution of lysine 110 by histidine, to provide a pH-sensitive positive charge at this position in the neutral pH range, exhibited only a moderate 25-fold increase in Km and a normal kcat at pH 6.0, whereas at pH 8.5 the Km for NADH rose to 238 microM with a decrease of 45% over unmodified enzyme in the kcat. A similar pH sensitivity in the inhibition constant for adenosine diphosphate ribose, lacking only the nicotinamide moiety of NADH, emphasizes the crucial role of the positive charge at this locus and is consistent with charge-pairing of lysine 110 with the pyrophosphate group of NADH or adenosine diphosphate ribose.

Characterization of lysyl residues of NADH-cytochrome b5 reductase implicated in charge-pairing with active-site carboxyl residues of cytochrome b5 by site-directed mutagenesis of an expression vector for the flavoprotein.

An expression vector for bovine NADH-cytochrome b5 reductase was constructed from two DNA fragments that were derived from beef liver poly(A+) RNA using the polymerase chain reaction. Site-directed mutagenesis of the 3 lysine residues of the reductase, previously implicated in the formation of active-site charge pairs with carboxylate residues of cytochrome b5, was then used to obtain the purified catalytic domains of flavoproteins modified at each of these sites. The observed marked decreases in catalytic efficiencies of substitutions of a negative charge at the normally positively charged residues with the catalytic domain of cytochrome b5 are consistent with their participation in the formation of charge pairs with carboxylate groups of the hemeprotein to optimize rapid electron transfer from the reductase flavin to the heme of the cytochrome.

7,12-Dimethylbenz[a]anthracene adduct formation with Syrian hamster cheek pouch epithelial DNA: in vitro studies in organ explant culture.

Studies examined the binding of radiolabeled 7,12-dimethylbenz[a]anthracene (DMBA) to epithelial DNA of hamster cheek pouch (HCP) maintained in organ explant culture. Adduct formation was studied as functions of [3H]DMBA dose, of the time after single [3H]DMBA applications, and of the route by which the DMBA was administered--either topically or in the culture media. Total DMBA-DNA adduct formation [total binding index (TBI)] was determined by DNA-bound 3H activity, and qualitative binding characteristics were further studied by high-pressure liquid chromatography. [3H]DMBA was applied either in the culture media at concentrations of 0.005-0.5 micrograms/ml or topically in mineral oil or ethanol in doses of 0.005-0.5 micrograms to each tissue fragment. Histopathologic changes in DMBA-treated HCP fragments included substantial aberrations in maturation of cornified and keratin layers and focal squamatization and dysplasia of the basal epithelium--considerable tissue necrosis was encountered in the high-DMBA-dose groups. Dose-response data were qualitatively similar among treatment types, with the greatest TBIs in topical ethanol groups and the lowest TBIs in culture medium groups. Kinetics of adduct formation and removal showed a rapid increase in TBIs to peak values at 24-72 hours followed by a biphasic decrease in TBIs, which leveled off at 7%-20% of peak values at 120-240 hours. Chromatographic analyses of selected samples at various times from all treatment groups showed three major peaks that are likely to be the same 1,2,3,4-tetrahydro-3,4-dihydroxy-1, 2-oxide-deoxyribonucleoside adducts observed in other rodent in vivo and cell culture systems. These results are consistent with those of other laboratories studying DMBA-DNA interactions and suggest that in vitro studies of DMBA-treated HCP explants are useful in studying the molecular nature of DMBA-DNA interactions in oral mucosal carcinogenesis.

DNA adduct formation by 7,12-dimethylbenz(a)anthracene in Syrian hamster cheek pouch epithelium in vivo.

Studies examined the in vivo binding of radiolabeled 7,12-dimethylbenz(a)anthracene (DMBA) to hamster cheek pouch epithelial DNA. Adduct formation was studied as functions of [3H]DMBA dose and of the time after single [3H]DMBA applications in mineral oil. Total DMBA-DNA adduct formation was determined by DNA-bound 3H activity, and qualitative binding characteristics were further studied by high-pressure liquid chromatography. Adduct formation 24 h after single [3H]DMBA applications rapidly increased from DMBA concentrations of 0.05-5.0 micrograms. While binding also increased from DMBA concentrations of 5.0-50.0 micrograms, the variability in adduct formation at 50.0 micrograms was considerable. Adduct formation following single 5.0-micrograms [3H]DMBA applications rose slowly to a peak value of 76 pmol DMBA/mg DNA at 36 h. This level decreased very slowly in a biphasic manner through 240 h, at which time the adduct levels were 23% of maximum. Adduct levels of 1.5 pmol/mg DNA were measured as late as 5 wk after a single 5.0-micrograms [3H]DMBA application. Chromatographic analyses of the 24-, 36-, and 96-, and 240-h samples showed three major peaks which are likely to be 1,2,3,4-tetrahydro-3,4-dihydroxy-1,2-oxide-deoxyribonucleoside adducts. While these analyses were limited by the small amounts of radioactivity which could be retrieved from [3H]DMBA-treated pouches, the study suggests that the DMBA-induced hamster cheek pouch carcinoma may be useful in some molecular in vivo studies of chemical-DNA interactions in carcinogenesis.

Low-level X-radiation effects on functional vascular changes in Syrian hamster cheek pouch epithelium during hydrocarbon carcinogenesis.

Effects of repeated low-level X radiation on functional microvascular changes in hamster cheek pouch epithelium during and following carcinogenesis by 7,12-dimethylbenz[a]anthracene (DMBA) were studied. Prior studies showed enhancement of such carcinogenesis by repeated 20 rad head and neck X-radiation exposures, and it was proposed that one possible mechanism was radiogenic alteration of the functional microvasculature in a manner which favored subsequent tumor development. Hamsters were treated with either radiation, DMBA, radiation + DMBA, or no treatment. Animals were sacrificed at 3-week intervals from 0 to 39 weeks after treatments began. Pouch vascular volume and permeability changes were studied by fractional distributions of radiotracers and were analyzed by a variety of statistical methods which explored the vascular parameters, treatment types, elapsed time, presence of the carcinogen, and histopathologic changes. All treatments resulted in significant changes in vascular volume with time, while only DMBA treatments alone resulted in significant changes in vascular permeability with time. Prior to the appearances of frank neoplasms, volumetric changes in DMBA only and radiation only groups were similar, while volume changes in DMBA + radiation groups increased slowly to a peak later than in other groups and then declined steadily to levels similar to the radiation only group. As in prior studies, there were significant vascular volume differences between DMBA and DMBA + radiation groups of tumor-bearing cheek pouches. DMBA maxima were significantly higher than those of DMBA + radiation. Radiation significantly affected DMBA-associated vascular volume and permeability changes during carcinogenesis. Several possible explanations for the relationship of these changes to the enhancement of DMBA carcinogenesis include: radiation blocking normal capillary proliferative and/or dilatory responses to inflammation secondary to neoplastic changes; radiation-induced focal increases in the pericapillary connective tissue histohematic barrier, stimulating angiogenesis but reducing nutrient diffusion; radiation exposures sensitizing vascular endothelium to subsequent angiogenic stimulation from premalignant tissues; DMBA vascular and epithelial effects partially or completely blocking radiation effects on epithelial and/or endothelial cells; and radiation damage to vessel walls partially or fully inhibiting normal physiologic mechanisms of repairing DMBA damage to the vessels.

Hemodynamic monitoring and vasodilator therapy in acute left ventricular failure. Preliminary report on a new vasodilator, tolmesoxide.

Data, obtained by invasive hemodynamic monitoring, are now routinely available in our Intensive Care Unit, and are processed by means of a small calculator to a cardiovascular profile, including O2 transport/consumption, vascular resistance and ventricular work values. Patients with clinically persistent shock states, after 12-24 hours optimal classical cardiovascular therapy, are invasively studied. Vasodiltor therapy frequently improves not only hemodynmic parameters, but also oxygen delivery to the tissues. Tolmesoxide, a new peripheal vasodilator, has been used in eleven patients. Preliminary results are discussed.