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Errors in predicting functional capacity for postmyocardial infarction patients using a modified Bruce protocol.

Clinically, the modified Bruce protocol is widely used to predict functional capacity in postmyocardial infarction (post-MI) patients. However, it has been suggested that post-MI patients have lower oxygen uptakes for standard workloads. In order to study this, we measured oxygen uptake (VO2) and venous blood lactic acid concentration in 12 post-MI patients and 12 normal male subjects during a modified Bruce treadmill protocol. During the first four stages of the protocol, mean oxygen uptake was significantly lower (1.0 to 6.2 cc X kg-1 X min-1; p less than 0.001) for the post-MI patients than for the normal male subjects. Venous blood lactic acid concentrations were different only at stage 4. However, a higher respiratory exchange ratio was observed for post-MI patients (p less than 0.001 at stage 3), suggesting an increased anaerobic metabolism and adequate buffering of lactic acid. The post-MI patient's measured VO2 for three stages of the protocol ranged from 1.8 to 7.3 cc X kg-1 X min-1 lower than the Bruce prediction for cardiac patients. In addition, maximal measured VO2 for the post-MI patients ranged from 3.7 to 11.2 cc X kg-1 X min-1 lower than predicted VO2 derived from the normal subjects. These data suggest myocardial damage may slow oxygen uptake kinetics, thus increasing the oxygen deficit at standard workloads during progressive exercise. An increased respiratory exchange ratio secondary to the buffering of lactic acid suggests that anaerobic metabolism may compensate for this oxygen deficit.

Coronary Disease↗

Usefulness of a branching treadmill protocol for evaluation of cardiac functional capacity.

A branching treadmill protocol was designed to measure functional capacity in patients with low work capacity and varying ability to walk at speeds used in traditional protocols. A comfortable walking pace is first selected (2.0 to 3.5 mph, 0.25 mph increments) and the workload is then increased every 2 minutes in 1 MET increments (a multiple of the resting oxygen uptake [1 MET = 3.5 ml O2/kg/min]) by adjusting grade. Nine trained (maximal MET = 7.6 +/- 1.6, mean +/- standard deviation) male subjects (age 59 +/- 7 years) with previous myocardial infarction and 9 trained (maximal MET = 11.7 +/- 2.5) male control subjects (age 56 +/- 8 years) completed submaximal and maximal workloads without handrail support. The measured oxygen consumption, volume of oxygen in ml/kg/min (VO2), was compared with the predicted VO2 cost of treadmill walking calculated from speed and grade. A linear regression analysis of predicted versus measured VO2 was performed. There were no significant differences between myocardial infarction and control regression lines. Therefore, a simplified prediction equation for estimated VO2 in myocardial infarction and control subjects is proposed. Overall VO2 prediction = 1.61 + 0.99 x. The main advantage of the branching protocol format is the selection of a stable, brisk walking pace compatible with age and gait, which may improve mechanical efficiency through impedance matching. The protocol is adaptable enough in design so that most patients can complete the exercise test without use of handrails, which is essential for an accurate estimate of VO2 from treadmill speed and grade.

Aged↗

An endoscopic biopsy protocol can differentiate high-grade dysplasia from early adenocarcinoma in Barrett's esophagus.

BACKGROUND: Surveying vs. performing resection in patients with high-grade dysplasia in Barrett's esophagus is debated because of concern about the accuracy of endoscopic biopsy diagnosis. The aim of this study was to investigate the accuracy of an endoscopic biopsy protocol in patients with neoplastic abnormalities in Barrett's epithelium without obvious esophageal cancer. METHODS: Preoperative and postoperative diagnoses in 28 patients who underwent surgery for high-grade dysplasia or early adenocarcinoma in Barrett's esophagus and compared them with 22 other patients with high-grade dysplasia who were maintained under prospective endoscopic surveillance. All 50 patients lacked gross lesions to suggest esophageal cancer. The endoscopic protocol involved rigorous, systematic acquisition of multiple, large biopsy samples. RESULTS: Overall, 64% of patients had minimal but distinct endoscopic abnormalities that were targeted for biopsies. High-grade dysplasia alone (7 patients) was differentiated from early adenocarcinoma (19 patients). Two patients with preoperative diagnoses of intramucosal adenocarcinoma had high-grade dysplasia in their resection specimens. CONCLUSIONS: This endoscopic protocol accurately detects and differentiates high-grade dysplasia from early adenocarcinoma in Barrett's esophagus. Patients with high-grade dysplasia alone in Barrett's esophagus detected by such a protocol do not necessarily require surgical resection to rule out an undiagnosed adenocarcinoma; electing for surgery should be based on other clinical considerations.

Adenocarcinoma↗

Extended fitness of variable region primers by a novel PCR protocol.

In an attempt to isolate the variable region genes of five monoclonal natural autoantibodies, we failed to amplify all ten variable region cDNAs using a V region primer set and following conventional PCR protocols. A novel PCR procedure was established in which the reaction began with three cycles of low temperature annealing and chain extension using 5'-primer as the only primer present, and continuing with conventional PCR cycles following addition of 3'-primer. Combined with solid-phase cDNA synthesis on oligo(dT)cellulose, this new protocol rescued all failed PCR amplifications based on a conventional protocol, thereby extending the universal utility of the V region primers. A notable increase in amplification specificity was also achieved. Such a protocol could be useful in primer design and the amplification of non-antibody genes whose sequences are not well-understood.

Animals↗

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: study design.

The most commonly used genotoxicity assays for cultured mammalian cells are mammalian cell mutagenesis, chromosome aberrations/SCE, hepatocyte UDS, and cell transformation. Since their inception, protocols for these assays have been modified in various laboratories. It has been observed that minor but potentially significant method modifications frequently remain unpublished (Swierenga et al., 1983) but should be considered in the development of recommended protocols. The present study was undertaken to determine the current 'state of the art' for these tests. Detailed questionnaires on culture conditions and testing protocols for both stock and test cell populations were designed with the assistance of an international advisory committee and sent to all research and contract laboratories that could be readily identified in Canada, U.S.A. and Europe. Responses from 425 completed questionnaires were analyzed to determine the most commonly used approach and modifications for each procedural step. As expected, the results show a large degree of interlaboratory variation. Detailed protocols for conducting each assay have been prepared and include: stepwise instructions, precautionary measures and practical solutions to common problems associated with each assay; recipes for media and solutions; formulas for quantifying genotoxic responses; reference lists of related assays; guidelines for interpretation; and discussions of the applications, advantages and disadvantages of each test.

Animals↗

A protocol for determining the shape of the ventilatory response to hypoxia in humans.

The ventilatory response to isocapnic hypoxia is biphasic, which makes any experimental assessment of the relationship between the acute (peak) ventilatory response and the level of hypoxia difficult. This study explored whether one particular protocol could be useful for determining this relationship. The protocol consisted of exposing subjects to seven different levels of isocapnic hypoxia, each of which lasted 50 sec. In order to test whether the order of the hypoxic exposure had any effect on the outcome, the steps were performed both in increasing and decreasing severity of hypoxia, and the ventilatory responses compared. Twelve subjects were studied, and each test was repeated four times in each subject. PETCO2 was held at 2 mmHg above resting throughout. The ventilations obtained at the lowest level of PETO2 employed were clearly different between the two protocols. However, provided that these ventilations were excluded, no significant differences were present between the results from the ascending and descending exposures (ANOVA). This finding suggests that the rate of change of PO2 in these protocols was sufficiently slow for a full ventilatory response to develop, but also sufficiently fast to prevent significant ventilatory depression from occurring.

Adult↗

Endocardial infiltrates in human heart transplants: a serial biopsy analysis comparing four immunosuppression protocols.

Endocardial mononuclear cell infiltrates were studied in 2,350 consecutive biopsies from 172 patients over a period ranging from 4 to 16 months post cardiac transplantation. The patients, otherwise unselected, were equally subdivided into four groups based upon the specific type of maintenance immunosuppression used. This was to allow for comparison of the effects of four separate commonly used recipient immunosuppression protocols, which could potentially influence the characteristics of the infiltrates. With azathiaprine-corticosteroid immunosuppression, endocardial infiltrates in otherwise normal biopsies were exceedingly rare, very minor, and invariably unifocal. Mild and moderate rejection were associated with a highly significant stepwise increase in incidence, prominence, and multifocality of endocardial infiltrates. In contrast, with each of the three cyclosporine-based recipient immunosuppression protocols which were evaluated, approximately 15% of biopsies with no evidence of rejection were associated with endocardial infiltrates. There was a wide range of variation in the prominence of the endocardial infiltrates present. Multifocal infiltrates were frequently encountered, the incidence of which was exclusively dependent upon the specific cyclosporine-based immunosuppression protocol used. With mild and with moderate rejection there was a significant stepwise increase in overall biopsy incidence of all endocardial infiltrates in each of the three groups, although there was no variation in relative prominence of the infiltrates, or in incidence of multifocality when biopsies without rejection were compared. The presence of conspicuous vascular spaces within endocardial infiltrates and significant extension of the infiltrates into the adjacent myocardium, with or without associated myofiber necrosis, were characteristic features of the most prominent endocardial infiltrates in all three cyclosporine-based immunosuppression groups. This constellation of changes has sometimes been referred to as "Quilty" effect. The relative incidence with which these particular features were encountered in association with endocardial infiltrates did not vary with rejection category of the biopsies. This study has shown that the presence of all forms of endocardial infiltrates, in the absence of concomitant rejection, is a characteristic manifestation of cyclosporine-based recipient immunosuppression, regardless of the specific protocol and cyclosporine dosage schedule. Under azathiaprine-based immunosuppression, endocardial infiltrates are almost invariably associated with rejection. It is postulated that cyclosporine-related endocardial mononuclear cell infiltration, in the absence of overt rejection, may result from a low level alloimmune response secondary to fluctuations in cyclosporine drug levels or related factors, and that the incidence with these infiltrates occur can be augmented during acute rejection episodes when the strength of the recipient immune response is magnified.

Biopsy↗

Admission criteria for immunogerontological studies in man: the SENIEUR protocol.

Immunogerontological studies in man have often led to conflicting results. One of the main reasons is the selection of the subjects to be studied. Admission criteria such as "apparently healthy" or "without overt disease" seem insufficient to exclude underlying disease which might influence the immune system and thereby the results. In an attempt to solve this problem, the SENIEUR protocol described in this paper was developed by a working party in the framework of the EURAGE Concerted Action Programme on Ageing of the European Community. This protocol establishes strict admission criteria for immunogerontological studies in man based on clinical information and laboratory data, and it sets limits to pharmacological interference. The use of this protocol will lead to standardization between centers and also to a closer study of the influence of age as such on the immune system. These findings in the immunologically "optimally aged" can also serve as reference values for immunogerontological studies in subjects who do not meet the SENIEUR criteria. In this way the use of this protocol can contribute to the dissection of the influence of disease versus ageing on the immune system.

Adult↗

Necessity of the assessment of health status in human immunogerontological studies: evaluation of the SENIEUR protocol.

Disease is frequent in ageing, and the many conflicting results in studies of the ageing process can be due to the presence of factors such as underlying disease or the use of medication. For immunogerontology, a solution to this problem was initiated in 1984 by a working party of EURAGE, the European Community's Concerted Action Programme on Ageing and Diseases. A protocol defining strict admission criteria to studies of ageing, the SENIEUR protocol, was elaborated. This protocol intends to limit the influence of disease and/or medication and to standardize admission criteria to immunogerontological studies. In subjects fulfilling the SENIEUR criteria, we found less immunological defects with ageing than generally stated. This could mean that many studies performed in not-optimally healthy subjects describe defects that are not a consequence of the ageing process, but could be a result of underlying disease or of the influence of medication. For lymphocyte subsets, certain changes are only found in the comparison of SENIEUR groups of young and aged, while other changes are only found when non-healthy groups are compared. The occurrence of monoclonal gammopathies and autoantibodies was increased in ageing, but was also influenced by health status. Experience of other groups, and the objections against the protocol are discussed.

Adult↗

The effect of estrus synchronization scheme, injection protocol and large ovarian follicle on response to superovulation in beef heifers.

Two trials were conducted to examine the effects of estrus synchronization scheme, gonadotropin injection protocol and presence of a large ovarian follicle on response to superstimulation of follicular development and the ensuing superovulation. Estrus was synchronized with either a progestin compound (MGA) or by the use of a luteolytic agent (PGF). Superstimulation was induced with 280 mg equivalents of pFSH administered either by a single subcutaneous injection or by a series of 8 intramuscular injections over 4 d. Follicular development was followed for 5 d with real-time ultrasound, and the heifers were retrospectively classified as to the presence or absence of a large follicle (> or = 8 mm; morphologically dominant follicle) at the start of superstimulation. The 2 trials differed by season of the year and genetic origin of the heifers. In Trial I (20 heifers), the ovulation rate was influenced by the 3-way interaction of the synchronization scheme, injection protocol and morphologically dominant follicle (P = 0.05). The number of large follicles on Day 5 (Day 0 = day of start of superstimulation) and ovarian score (scale 1 to 5 based on extent of follicular development; 1 = least, 5 = most) on Day 5 were significantly correlated (P < 0.05) with ovulation rate. In Trial II (20 heifers), the ovulation rate, number of embryos recovered, number of transferable embryos and ovarian weights were all greater (P < 0.05 to P < 0.01) with the 8-injection protocol than the 1-injection protocol. The number of medium follicles (5 to 7 mm) on Days 2 and 3, number of large follicles (> or = 8 mm) on Days 3, 4 and 5 and ovarian scores on Days 4 and 5 were all significantly correlated (P < 0.05) with ovulation rate. In both trials, differences in follicle populations were not seen until Day 3 of the superstimulation procedure. Collectively, these trials do not provide strong support for a single injection of FSH, as used here, nor does it indicate a clear advantage for either MGA or PGF as a means of enhancing the ovulation rate or embryo quality.

Journal Article↗

Anterograde axonal tracing with the subunit B of cholera toxin: a highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains.

We report an improved immunohistochemical protocol for revealing anterograde axonal transport of the subunit B of cholera toxin (CTB) which stains axons and terminals in great detail, so that single axons can be followed over long distances and their arbors reconstructed in their entirety. Our modifications enhance the quality of staining mainly by increasing the penetration of the primary antibody in the tissue. The protocol can be modified to allow combination in alternate sections with tetramethylbenzidine (TMB) histochemical staining of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Using the protocol, we tested the performance of CTB as an anterograde tracer under two experimental paradigms which render other anterograde tracers less sensitive or unreliable: (1) labeling the entire retinofugal projection to the brain after injections into the vitreal chamber of the eye, and (2) labeling developing projections in the cortex and thalamus of early postnatal mammals. Qualitative comparisons were made with other tracers (Phaseolus vulgaris leucoagglutinin, dextran rhodamine, biotinylated dextran, free WGA, or WGA-HRP) that were used to label these same projections. From these observations it is clear that CTB, visualized with our protocol, provides more sensitive anterograde labeling of retinofugal projections as well as of axonal connections in the neonatal forebrain.

Animals↗

Micronucleus induction by azobenzene and 1,2-dibromo-3-chloropropane in the rat: evaluation of a triple-dose protocol.

A triple-dose protocol was evaluated in the rat bone-marrow micronucleus test using azobenzene and 1,2-dibromo-3-chloropropane (DBCP) as test compounds. Dosing with 250 mg/kg azobenzene over 3 consecutive days led to an accumulation of micronucleated polychromatic erythrocytes. The magnitude of the effect was the same as that obtained after a single dose of 750 mg/kg in a previous study. In the single-dose study the effect was only detected at the 48 h sampling time. With the triple-dose protocol the effect was apparent 24 h after the last dose--thus eliminating the need for more than one sampling time. In the case of DBCP, the positive effect observed after a single dose diminished with increasing number of doses for the low dose but plateaued for the high dose, accompanied by a marked cytotoxic effect. It is therefore concluded, that for one of the compounds, azobenzene, the triple-dose protocol provided an advantage whereas for the other, DBCP, it failed to do so and led to results which are rather difficult to interpret. Furthermore, the number of animals necessary to select an appropriate dose level appears to be higher than in the single-dose protocol.

Animals↗

Activity of the human carcinogens benzidine and 2-naphthylamine in triple- and single-dose mouse bone marrow micronucleus assays: results for a combined test protocol.

The activities of the human bladder carcinogens benzidine and 2-naphthylamine in the mouse bone marrow micronucleus assays using a limited test protocol (oral dosing to male mice, sampling 24 h later) have recently been established. As a contribution to the International Collaborative Study on the evaluation of the sensitivity of the triple-dose micronucleus test protocol it was decided to re-evaluate benzidine and 2-naphthylamine using a combined triple- and single-dose test protocol. Benzidine gave a clear positive response in male mice 24 h after 3 daily doses of 150 and 300 mg/kg. A single dose of 900 mg/kg of benzidine gave a weaker response 24 h after dosing. In the case of 2-naphthylamine a stronger positive response was observed 24 h after a single dose of 600 mg/kg as compared to 3 daily doses of 200 or 400 mg/kg. There was no significant difference in the increased positive response observed for a single dose of 30 mg/kg of cyclophosphamide compared with 3 successive daily doses of 10 mg/kg. Based on the present data the combined triple/single-dose micronucleus test protocol is strongly supported.

2-Naphthylamine↗

Effect of excess dietary retinoic acid on skin papilloma and carcinoma formation induced by a complete carcinogenesis protocol in female Sencar mice.

Previously, we have shown that dietary retinoic acid (RA) at pharmacological doses (30 micrograms/g of diet) inhibited the malignant conversion of skin papillomas to carcinomas induced by a two-stage carcinogenesis protocol with 7,12-dimethylbenz[a]anthracene as initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as promoter (De Luca et al., Carcinogenesis, 14 (1993) 539-542). The purpose of this study was to determine the effect of dietary RA on skin papilloma and carcinoma formation induced by a complete carcinogenesis protocol with repeated DMBA treatment in female Sencar mice. Mice at 3 weeks of age were weaned onto a diet containing either 3 (control) or 30 (excess) micrograms of RA/g of diet and treated topically with DMBA (25.5 micrograms) once per week for 20 weeks. Mice fed excess dietary RA did not significantly differ from control mice in the following parameters: body weight, survival rate, papilloma incidence, cumulative carcinoma incidence (19.4% versus 23.7%), carcinoma yield (0.19 versus 0.26 per mouse), carcinoma conversion efficiency (5.2% versus 3.9%), and average age of carcinoma development (22.7 +/- 4.7 versus 23.3 +/- 2.8 weeks). However, papilloma yield was decreased by about 50% (i.e. 3.7 versus 7.0 at week 20, P < 0.01) between weeks 17 and 22 of age by excess dietary RA treatment. Contrary to other routes of administration (i.e. topical and systemic) of RA (Verma et al., Cancer Res., 42 (1982) 3519-3525), excess dietary RA did not enhance skin tumor formation. In addition, excess dietary RA failed to inhibit malignant conversion of papillomas to carcinomas in the complete carcinogenesis protocol. Thus, the modulation of RA on skin papilloma and carcinoma formation is dependent on carcinogenesis protocol and route of RA administration.

9,10-Dimethyl-1,2-benzanthracene↗

Protocol variations and their influence on the final outcome: critical review of the split-course base of tongue study of the RTOG.

A critical review of the protocol variations in an RTOG prospective randomized multi-institutional clinical trial has been conducted. The objective of the review was to identify variables that might have affected the results of the study. Compliance with general guidelines and specific technical treatment parameters of the Split-Course Radiation Therapy Protocol for Carcinoma of the Base of the Tongue (RTOG #71-02) was analyzed. A +/- 5% variation of the time-dose-fractionation factors, using the NSD and TDF formulae and their respective protocol ratios (ratio between actual case NSD and TDF level and what the protocol recommended), has been defined as fully acceptable. Seventy-two percent of the 141 study cases were within this category. Minor variation has been defined as one resulting in a +/- 6% to +/- 15% deviation from the study values (15.3% of cases). Major and unacceptable variation was a deviation of +/- 16% or more from the research plan (12.7% of cases). After identifying the different variations and correlating them with the clinical results, we conclude that these deviations did not affect the final results to a significant or identifiable degree.

Clinical Trials as Topic↗

Multi-center trial with an in vitro NMR protocol. EEC Concerted Research Project.

The EEC Protocol for in vitro measurement of T1 and T2 presented in paper II of this series was tested in 15 centers on a variety of instruments. This article discusses the results of this protocol trial using biological samples (rat liver and thigh muscle) and two reference gel samples. Each reference gel was prepared as a single batch, dispensed into appropriate sample tubes and sent to all participants. Details of instrument types and operating conditions are given, along with measurement frequencies and temperatures and the results of precision testing. The relaxation time measurements from the gels for the trial are compared with temperature and frequency effects examined in independent centers under similar conditions. The relaxation time values for biological tissues are examined in the light of the scatter of such values in the general literature. To check the effects of data analysis methods on relaxation times, full relaxation data for one reference sample was provided by each group. This was re-analyzed in three centers using different methods; these results are compared with the values calculated by each group using its own method. In general, the results from different groups show good consistency with the reference values. The use of this Protocol has helped reduce scatter in results from biological samples and has provided information for improvement of individual operating conditions and improvement of the protocol to its final form.

Animals↗

Orthodontic treatment time in 2- and 4-premolar-extraction protocols.

INTRODUCTION: The purpose of this study was to compare the treatment times of complete Class II malocclusions treated with 2- and 4-premolar-extraction protocols. METHODS: Ninety-seven patients were selected and divided into 2 groups, according to the 2 extraction criteria. Group 1, treated with 2 maxillary premolar extractions, consisted of 49 patients (30 male, 19 female) with a mean age of 14.35 years. Group 2, treated with 4 premolar extractions, consisted of 48 patients (27 male, 21 female) with a mean age of 13.03 years. Treatment times of the groups were compared with the t test. RESULTS: Treatment times were significantly shorter with the 2-premolar-extraction protocol compared with the 4-premolar-extraction protocol. CONCLUSIONS: Treatment time will be shorter and the occlusal results more predictable with a 2-premolar-extraction protocol compared with 4 premolar extractions.

Adolescent↗

Comparison of intubation attempts and completion times before and after the initiation of a rapid sequence intubation protocol in an air medical transport program.

The purpose of this study was to test the hypothesis that the initiation of a rapid sequence intubation (RSI) protocol would decrease the number of intubation attempts and completion times. In the summer of 2001, an RSI protocol was implemented using succinylcholine and etomidate. Before the RSI protocol, patients were sedated with versed and fentanyl for intubation and then paralyzed with succinylcholine to complete intubation, if needed. Retrospectively reviewed patient transport charts yielded 70 patients in the pre-RSI group and 70 patients in the RSI group. The number of attempts in the RSI group significantly dropped in the pre-RSI group, and the time from medication administration to intubation statistically decreased. In this study, the implementation of an RSI protocol resulted in fewer intubation attempts and more rapid intubations.

Adult↗