Prospective multicenter evaluation of the pulmonary embolism rule-out criteria.

BACKGROUND: Over-investigation of low-risk patients with suspected pulmonary embolism (PE) represents a growing problem. The combination of gestalt estimate of low suspicion for PE, together with the PE rule-out criteria [PERC(-): age < 50 years, pulse < 100 beats min(-1), SaO(2) >or= 95%, no hemoptysis, no estrogen use, no surgery/trauma requiring hospitalization within 4 weeks, no prior venous thromboembolism (VTE), and no unilateral leg swelling], may reduce speculative testing for PE. We hypothesized that low suspicion and PERC(-) would predict a post-test probability of VTE(+) or death below 2.0%. METHODS: We enrolled outpatients with suspected PE in 13 emergency departments. Clinicians completed a 72-field, web-based data form at the time of test order. Low suspicion required a gestalt pretest probability estimate of <15%. The main outcome was the composite of image-proven VTE(+) or death from any cause within 45 days. RESULTS: We enrolled 8138 patients, 85% of whom had a chief complaint of either dyspnea or chest pain. Clinicians reported a low suspicion for PE, together with PERC(-), in 1666 patients (20%). At initial testing and within 45 days, 561 patients (6.9%, 95% confidence interval 6.5-7.6) were VTE(+), and 56 others died. Among the low suspicion and PERC(-) patients, 15 were VTE(+) and one other patient died, yielding a false-negative rate of 16/1666 (1.0%, 0.6-1.6%). As a diagnostic test, low suspicion and PERC(-) had a sensitivity of 97.4% (95.8-98.5%) and a specificity of 21.9% (21.0-22.9%). CONCLUSIONS: The combination of gestalt estimate of low suspicion for PE and PERC(-) reduces the probability of VTE to below 2% in about 20% of outpatients with suspected PE.

Isolating antigenotoxic components and cancer cell growth suppressors from agricultural by-products.

Commercial processing wastes or by-products of crops were found to be sources of antimutagens and human tumor cell growth suppressors. We developed a microplate method to measure genomic DNA damage in Chinese hamster ovary cells with a modified single cell gel electrophoresis (SCGE) assay. This allowed us to measure the repression of 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage by very small amounts of complex mixtures, fractions or individual chemicals isolated from agricultural by-products. We previously demonstrated that PCC, an ethanol extract of a commercial soybean processing by-product, repressed induced genomic DNA damage in mammalian cells. PCC was separated into a series of chemically defined fractions and two fractions (PCC70 and PCC100) repressed mutagen-induced damage. Of the isoflavones isolated from soybean fraction PCC70, daidzein expressed antigenotoxic activity, however, genistin and genistein enhanced DNA damage. An antigenotoxic response also was observed with a fraction isolated from corn distillate solids (CDS40). We developed a microplate assay to measure the suppression of the growth rate of human cancer cells in which the cytostatic/cytotoxic status at each concentration of the test sample was quantitatively determined. Genistein, genistin, daidzein and daidzin isolated from soybean fraction PCC70 expressed a wide range of growth suppression of HT-29 human colon cancer cells. The biological assays were integrated with, and directed, the separation and analytical chemistry component of this project. Compounds were purified from biologically active fractions and the structure of individual chemicals was determined with analytical HPLC and LC-mass spectroscopy (LC-MS). This research may lead to the isolation of novel chemoprotectants from agronomic commercial processing products and by-products.

Diagnostic accuracy of a bedside D-dimer assay and alveolar dead-space measurement for rapid exclusion of pulmonary embolism: a multicenter study.

CONTEXT: A previous study suggested that the combination of a normal D-dimer assay and normal alveolar dead-space fraction is a highly sensitive screening test for pulmonary embolism (PE). OBJECTIVE: To determine if the combination of a normal alveolar dead-space fraction (volume of alveolar dead space/tidal volume

A comparison of DNA repair using the comet assay in tobacco seedlings after exposure to alkylating agents or ionizing radiation.

We employed single cell gel electrophoresis to analyze the kinetics of DNA repair in nuclei isolated from tobacco plants exposed to ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU) and gamma-radiation. DNA repair was measured as the reduction of the tail moment values as a function of time after the mutagen treatment ended. DNA damage in leaf nuclei of EMS-or ENU-treated tobacco plants persisted over a 72h recovery period. However, a reduction of the SCGE tail moment values in nuclei isolated from leaves was observed over a 4-week period of recovery. Newly emerged leaves expressed a lower level of DNA damage due to more efficient repair and/or dilution of initial DNA lesions during cell division. After 24h recovery, leaf nuclei from cells exposed to 20 or 40Gy of gamma-radiation expressed complete DNA repair. These data indicate that DNA lesions induced by alkylating agents are not readily repaired and persist beyond 4 weeks. Enzymes necessary to repair gamma-induced DNA lesions are fully functional in non-replicating leaf cells and single and double strand breaks are rapidly repaired.

Characterization and antimutagenic activity of soybean saponins.

An extract was prepared from a commercial soybean-processing by-product (soybean molasses) and was fractionated into purified chemical components. In previous work, this extract (phytochemical concentrate, PCC) repressed induced genomic DNA damage, whole cell clastogenicity and point mutation in cultured mammalian cells. In the current study, a chemical fraction was isolated from PCC using preparative high-performance liquid chromatography (HPLC). This fraction, PCC100, repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage in Chinese hamster ovary (CHO) cells as measured by single cell gel electrophoresis (alkaline Comet assay). Using liquid chromatography-electrospray ionization-mass spectroscopy and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, PCC100 was shown to consist of a mixture of group B soyasaponins and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) soyasaponins. These include soyasaponins I, II, III, IV, V, Be, betag, betaa, gammag and gammaa. Purified soyasapogenol B aglycone prepared from fraction PCC100 demonstrated significant antigenotoxic activity against 2AAAF. To our knowledge, these data demonstrate for the first time the antimutagenic activity of soybean saponins in mammalian cells.

Collaboration among emergency medicine physician researchers and statisticians: resources and attitudes.

We examined the statistical resources within emergency medicine residency programs, and the attitudes of emergency medicine physician researchers toward activities wherein collaboration with a statistician is useful. Anonymous surveys were mailed to 104 emergency medicine physician researchers (1/program). Sixty-four (62%) responses were analyzed. Sixty-seven percent of respondents were their program's research director. Their highest level of statistical training was self-taught/nondegree course work for 88% of respondents. Forty-two percent said they were the person used most often by their program for statistical expertise. One-quarter of programs employed a full-time statistician. Collaboration among researchers and statisticians was considered sometimes or always useful for protocol development (aims 84%, design 99%, outcomes 99%, procedures 73%, sampling 97%, inclusion criteria 93%, number of subjects 100%); data entry 73%; statistical analysis 100%; and manuscript preparation 86%. Although most emergency medicine residencies lacked statistical resources within their program, physician researchers expressed positive attitudes toward collaboration with a statistician for all aspects of research.

Studies on the genotoxic effects of aminophenazines using two cellular models and three different methods.

This paper presents studies on the genotoxicity of two aminophenazines: 2,3-diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP). The genotoxic activities of these compounds were evaluated with human lymphocytes using the alkaline single cell gel electrophoresis (SCGE) assay and two cytogenetic assays (chromosome aberrations (CA) and sister chromatid exchange (SCE) analysis). Results show that these chemicals elicited an increase in DNA and chromosomal damage under the studied ranges of concentration. Concentration-response curves were similar and there was a positive correlation between the damage observed at the DNA and chromosomal levels. DAP was more genotoxic than AHP and this agreed with the genotoxic potencies reported in bacterial systems.

Comparison of DNA damage in plants as measured by single cell gel electrophoresis and somatic leaf mutations induced by monofunctional alkylating agents.

The use of single cell gel electrophoresis (SCGE) has recently been applied to plant systems. We optimized the experimental conditions for SCGE analysis using nuclei isolated from different tissues of intact plants. Concentration-response curves of genomic DNA migration were analyzed in intact plants treated with the monofunctional alkylating agents ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), and N-methyl-N-nitrosourea (MNU). These data were used to calibrate SCGE tail moment values to induced somatic mutation in plant leaves. We used a genotoxicity index to compare genomic DNA damage and the induction of somatic mutation in the leaf tissues. The rank order of the genotoxic potency of these alkylating agents assayed by SCGE was MNU >> MMS > ENU > EMS. The rank order for the mutagenic potency of these agents was MNU >> ENU congruent with MMS > EMS. The data demonstrate the utility of SCGE analysis in plant systems. The use of SCGE will permit a larger range of plants for use as in situ environmental monitors. Also, this approach may be used to search for crop plant germplasm accessions with enhanced genomic stability. We investigated whether the intragenomic distributions of DNA damage induced by these alkylating agents were uniform and random. When a plot of the ratio of the %tail DNA and tail length versus the concentration of the test mutagen was generated, the induced SCGE data deviated from a random distribution of genomic DNA damage.

Antimutagenic activity of chemical fractions isolated from a commercial soybean processing by-product.

Commercial products of agronomic crop plants may become a reliable and inexpensive source of phytonutrients, such as antimutagenic food supplements. We previously demonstrated that PCC, an ethanol extract of a commercial soybean processing by-product, was able to repress induced genomic DNA damage, whole cell clastogenicity, and point mutation in mammalian cells. In this paper we separated PCC into a series of chemically defined fractions and determined their ability to repress induced mutagenic damage in Chinese hamster lung cells, Chinese hamster ovary cells and human lymphocytes. Fraction PCC70 (PCC 70% methanol eluate) contained the flavonoids from PCC and daidzin and genistin repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage measured with single cell gel electrophoresis. Genistein, however, enhanced the genotoxic impact of 2AAAF. Fraction PCC100 (PCC 100% methanol eluate) had the greatest level of antigenotoxic activity against 2AAAF in CHO cells and repressed the genotoxic capacity of the dietary carcinogen 2-amino-3-methylimidazo-(4,5-f)quinoline (IQ) in human lymphocytes. These data indicate that commercial soybean products and by-products may be a source of chemoprotective food additives.

Pentachlorophenol-mediated mutagenic synergy with aromatic amines in Salmonella typhimurium.

Pentachlorophenol (PCP), a widely used pesticide, enhanced the mutagenic potency of plant- or mammalian-activated 2-aminofluorene (2AF) as well as the direct-acting mutagen 2-acetoxyacetylaminofluorene (2AAAF) when assayed with specific Salmonella typhimurium strains. With 2AF the mutagenic synergy was observed in strains YG1024, TA1538, and MP153. With 2AAAF the PCP-mediated synergy was observed with these strains and with strain TA98/1,8-DNP6. The synergy was dependent upon the presence of an activated N-acetoxy functional group and was only expressed at the hisD3052 allele and not at the hisG46 allele. Spectrophotometric analysis demonstrated that the rate of degradation of 2AAAF was reduced in the presence of PCP in phosphate buffer or with S. typhimurium cytosol and thus PCP may be affecting the stability of the N-acetoxy group of activated aromatic amines.

Single cell gel electrophoresis analysis of genomic damage induced by ethyl methanesulfonate in cultured tobacco cells.

A procedure for employing cultured tobacco cells (line TX1) in the SCGE assay was developed. The effect on DNA migration was studied in control and EMS-treated cells at different stages in their growth curve. The experimental parameters of treatment time and the unwinding time were analyzed in TX1 cells. With EMS in a concentration range from 0 to 30 mM the average median (+/-S.E.) tail moments ranged from 2.71+/-0.24 microm for the negative controls and increased in a direct concentration dependent manner to 57.89+/-4.13 for cells treated with 30 mM EMS. Nuclei isolated from TX1 cells and treated with EMS had a similar sensitivity as TX1 cells after EMS treatment. The plant cells express similar concentration-response curves for EMS as reported with mammalian (CHO) cells. This plant cell SCGE assay may prove to be a useful tool for the study of agricultural chemicals in specific plant cell types, to compare the response of mutagens in plant and animal cells and for basic research in genetic toxicology and DNA repair in plants.

A computer model of gas exchange during cardiopulmonary resuscitation.

This paper presents a computer model of gas exchange during cardiopulmonary resuscitation (CPR) that permits independent adjustment of inspired air content (16% O2 and 4.5% CO2 present in mouth-to-mouth (MTM) ventilation or ambient air), shunt, deadspace, diffusion impairment, cardiac output, and ventilation. The model contains 15500 acini, each with its own blood supply. Gas exchange occurs at each perfused and ventilated acinus. Arterial P(O2) and P(CO2) are calculated from the summed arterial blood flow using standard formulae. The model and simulations show that MTM ventilation provides inadequate oxygenation when the victim is at high altitude or has diffusion impairment. They also show that analysis of inspired and expired gas concentrations to measure gas exchange primarily measures wash in and wash out of gas when cardiac output is low and that this explains the negative oxygen consumption and carbon dioxide production measured in a previous study.

The use of single cell gel electrophoresis and flow cytometry to identify antimutagens from commercial soybean by-products.

Single cell gel electrophoresis (alkaline Comet assay) and flow cytometric methods were combined into an assay that enables the analysis of direct DNA damage and longer-term whole cell clastogenicity in mammalian cells. We employed these techniques to analyze the antimutagenic activity of by-products of commercial soybean processing. At a concentration of 1 mg/ml, the soybean molasses by-product was found to repress 66% of the mutagenic capacity of the direct-acting mutagen 2-acetoxyacetylaminofluorene (2AAAF) in Chinese hamster lung (CHL) cells. At a concentration of 50 microg/ml, fraction PCC (an ethanol extract of soybean molasses) repressed 70% of the genotoxic potency of 500 nM 2AAAF as measured by the Comet assay. Fraction PCC was also effective in protecting CHL cells from 2AAAF-induced clastogenic damage. Using a forward mutation assay in Chinese hamster ovary cells (line AS52), PCC protected the cells against 2AAAF-induced cytotoxicity and point mutation at a specific gene target. These data indicate that agronomic crops such as soybean may yield a wealth of commercially available antimutagenic agents that may be suitable as chemoprotective food supplements.

Induction of somatic DNA damage as measured by single cell gel electrophoresis and point mutation in leaves of tobacco plants.

The induction and measurement of DNA damage in nuclei of plant tissues is a new area of study with the alkaline single cell gel electrophoresis/comet assay. Methods to isolate plant cell nuclei cause high levels of DNA damage which are detected by the comet assay. We developed a method to isolate nuclei from leaf tissue of Nicotiana tabacum (a1+/a1; a2+/a2) in a modified Sörensen buffer that resulted in constant, low tail moment values for the negative controls. After treating intact tobacco plants with 1-8 mM ethyl methanesulfonate (EMS) we obtained a direct concentration-response with an average median tail moment of 65.9+/-4.4 micro(m) for plants exposed to the highest EMS concentration as compared to the median control tail moment value of 4.1+/-0.8. We found that the highest resolution was obtained with electrophoretic conditions of 0.74 V/cm at 300 mA for 20 min. Multiple leaves could be analyzed per plant within each treatment group and the tail moments were not significantly different. Tobacco seedlings were treated with EMS in the same manner as used for the comet assay and mutations were induced in the leaf primordia. The mean mutant frequency for the control was 1.46+/-0.20 mutant sectors/leaf. The mutant frequency increased in a concentration dependent manner; the mutant frequency induced by 8 mM EMS was 37.89+/-2.37 mutant sectors/leaf. The comet tail moment values and the leaf mutant frequency were highly correlated (r=0.98). The genetic response factor was calculated by the ratio of the difference in the response within the linear portion of each concentration-response curve divided by the slope of the curve. The genetic response factor for the tail moment was 7.82 while the value for mutation induction was 7.76. In this paper we describe a sensitive method with high resolution to apply the alkaline comet assay to plant leaves. The comet assay response was compared to that of induced point mutation. With this sensitive method for nuclei isolation from plant leaves, the alkaline SCGE assay could be incorporated into in situ plant environmental monitoring.

Serum creatine kinase is an unreliable predictor of ectopic pregnancy.

OBJECTIVE: To compare serum creatine kinase (CK) values in patients with ectopic pregnancy vs patients with threatened miscarriage or normal pregnancy. METHODS: An observational case-control study was performed at an urban teaching hospital. Pregnant women with a quantitative beta-hCG obtained for suspicion of ectopic pregnancy were evaluated. Excluded were cases with recent trauma, i.m. injections, surgery, or history of heart, liver, or muscle disease. The serum beta-hCG and CK values were recorded and compared between groups with 1-way ANOVA and Tukey's multiple comparison procedure at the overall 0.05 level. RESULTS: The 15 ectopic, 28 threatened miscarriage, and 21 normal pregnancy cases were of similar gestational ages (p = 0.2), ranging from 3 to 12 weeks. Although the CK values for ectopic pregnancy (88.8 +/- 33.6 IU/L) exceeded those for threatened miscarriage (65.9 +/- 59.0 IU/L) and normal pregnancy (56.0 +/- 38.1 U/L) (p = 0.02), there was significant overlap between groups. CK values were at or above a cutoff of 74 IU/L in 80% (95% confidence interval: 52-96%) of ectopic pregnancies, 25% (11-45%) of threatened miscarriages, and 14% (3-36%) of normal pregnancies. CONCLUSIONS: Although the ectopic pregnancy population is characterized by a higher mean CK than are patients with threatened miscarriage or a normal pregnancy, a significant overlap in CK values makes use of this serum marker unreliable for detecting ectopic pregnancy.

The false-positive rate of thoracic outlet syndrome shoulder maneuvers in healthy subjects.

OBJECTIVE: To estimate the incidence of false-positive findings of thoracic outlet syndrome (TOS) shoulder maneuvers, Adson's test (AT), costoclavicular maneuver (CCM), elevated arm stress test (EAST), and supraclavicular pressure (SCP) in healthy subjects. METHODS: A cross-sectional, observational study was performed in a medical school and affiliated emergency medicine residency program setting. Participants included healthy adult volunteers without symptoms of TOS. The shoulder maneuvers AT, CCM, EAST, and SCP were performed in randomized order for 30 sec, 30 sec, 3 min, and 30 sec, respectively. Pulse quality and the presence and timing of pain or paresthesias were assessed. RESULTS: 53 subjects were enrolled, including 27 women, aged 29.7 +/- 6.4 years (range 21-58 years). AT, CCM, EAST, and SCP resulted in an altered pulse in 11%, 11%, 62%, and 21%; pain in 0%, 0%, 21%, and 2%; and paresthesias in 11%, 15%, 36%, and 15% of cases, respectively. The following outcomes had reasonable false-positive rates (upper 95% confidence limit): pain with the AT (7%), CCM (7%), SCP (10%), or any 2 TOS shoulder maneuvers (10%); discontinuing the EAST because of symptoms (16%); or any symptom with 3 (13%) or 4 (7%) TOS shoulder maneuvers. CONCLUSIONS: In a study of TOS shoulder maneuvers in healthy subjects, the outcomes of pulse alteration or paresthesias were unreliable in general. However, TOS shoulder maneuvers have reasonably low false-positive rates when a positive outcome is defined as pain after AT, CCM, or SCP; discontinuation of the EAST secondary to pain; pain in the same arm with > or =2 maneuvers; or any symptom in the same arm with > or =3 maneuvers.

The International Comet Assay Workshop.

The Comet (single cell gel electrophoresis) assay primarily measures DNA strand breakage in single cells (Singh et al., 1988). Briefly, cells are suspended in low melting point agarose on a microscope slide. The slides are put in lysing buffer to allow the DNA to unwind and then in electrophoresis buffer. During electrophoresis the broken DNA moves towards the anode forming a Comet tail, with the greater the extent of damage, the greater the tail. Assays can be conducted under neutral or alkaline (> pH 13) conditions. Double-strand breaks are measured under neutral conditions and single-strand breaks under alkaline conditions, where abasic sites and other alkali-labile sites or intermediates in base or nucleotide excision repair can also be detected. There are several good reviews concerning the assay (McKelvey-Martin et al., 1993; Fairbairn et al., 1995; Tice, 1995).