Comparative evaluation of whole blood D-dimer test to plasma D-dimer test for diagnosis of disseminated intravascular coagulation

Three rapid D-dimer test methods were compared for the diagnosis of acute disseminated intravascular coagulation (DIC). These were (a) SimpliRED, an autologous red cell agglutination assay. (b) DIMERTEST latex agglutination assay, containing monoclonal antibody DD-3B6/22(6), and (c) D-DI latex agglutination assay containing mouse anti-human D-dimer monoclonal antibodies. The D-DI latex method having higher sensitivity (100%) and specificity (81%) in clinically acute DIC was postulated as the gold standard and compared with the other two methods. The results suggest that D-DI latex agglutination assay containing mouse anti-human D-Dimer monoclonal antibodies are the better assay methods amongst all the three kits analyzed. It is advisable to look for the nature of the antibody used to coat the latex particles in plasma based kits. In emergency setting RBC kits may be of some use as rapid diagnosis is advantageous.     

Murine models for natural rubber latex allergy assessment

Murine models provide a powerful tool in the investigation of latex allergy and the development of intervention strategies. The immune responses to protein allergens of mice and humans are similar but differences related to the roles of IgE and IgG must be recognized. Mice have been shown to mount a dose and time-dependent IgE response to latex proteins following topical, respiratory, and subcutaneous exposures. Methods are available to evaluate cutaneous and respiratory responses to latex challenge in sensitized animals. These models have been used to investigate the role of route of exposure on the development of latex allergy and to provide a means for investigating the contribution of individual proteins to adverse respiratory and dermal responses. These models provide a mechanism for the evaluation of new technologies aimed at reducing the allergenicity of latex products, and for testing for the potential for cross-reactivity to new allergens in previously sensitized individuals. Murine models may also provide a method for testing immunotherapy strategies prior to initiating human trials.     

Permeability to phi chi 174 bacteriophages in polyolephin membrane condoms

Membranes used for the manufacture of condoms eventually can develop tiny pores, thereby decreasing dramatically their effectiveness as a physical barrier against the transmission of infectious agents. A technique was designed that was based on the ability of bacteriophage viruses to trespass membranes and to infect certain bacteria species, and then developing lysis plaques in the colonies of the host bacteria. The effectiveness of 60 polyolefin condoms in preventing the diffusion of the bacteriophage phi chi 174(ATCC13706-B1), 27 nm diameter, was compared to 20 latex condoms. Physiological conditions such as pressure, pH, superficial tension, length, time of exposure and viral titre were simulated. A pressurization system was designed, in which compressed air was injected simultaneously to ten condoms. Four of the 60 polyolefin condoms and one of the 20 latex condoms were permeable to the virus. Therefore, at least 93% of the condoms evaluated were able to contain the virus. The difference in permeability between the two types of membranes was not statistically significant (P = 0.79).     

Photochemical genotoxicity: principles and test methods. Report of a GUM task force

In recent years, assessing the photogenotoxic potential of a compound became an issue for certain drugs and cosmetical products. Therefore, existing methods performed according to international guidelines (e.g. OECD guidelines) were adapted to the use of concurrent UV-visible (UV-Vis) light irradiation for the assessment of photomutagenicity/photogenotoxicity. In this review, photobiological bases of the processes occurring in the cell after irradiation with UV- and/or visible (vis)-light as well as a compilation of testing methods is presented. Methods comprise cell free investigations on naked DNA and in vitro methods, such as the photo-Ames test, the photo-HPRT/photo-mouse lymphoma assay (MLA), the photo-micronucleus test (MNT), the photo-chromosomal aberration test (CA) and the photo-Comet assay. A compilation of the currently available international literature of compounds tested on photogenotoxicity is given for each method. The state of the art of photogenotoxicity testing as well as the rational for testing are outlined in relation to the recommendations reached in expert working groups at different international meetings and to regulatory guidance papers. Finally, photogenotoxicity testing as predictor of photocarcinogenicity and in the light of risk assessment is discussed.     

Latex and vinyl nonsterile examination gloves: status report on laboratory evaluation of defects by physical and biological methods.

We have reported previously (H. R. Kotilainen, J. P. Brinker, J. L. Avato, and N. M. Gantz, Arch. Intern. Med. 149:2749-2753, 1989) that the quality of nonsterile examination gloves available for clinical use may be extremely variable. In view of the concern over human immunodeficiency virus and hepatitis B virus transmission to health care workers, the continuing variability of gloves available for use, and the need for a simple and safe test, we have evaluated 2,500 vinyl (five brands) and 2,000 latex (four brands) gloves by the 300-ml and the newly proposed 1,000-ml water tests and for permeability to herpes simplex virus type 1 and poliovirus type 1, respectively. While all 300-ml watertight gloves were unlikely to leak herpes simplex virus type 1 (1.3% vinyl; 0.5% latex), poliovirus was recovered much more frequently (8.9% vinyl, 6.1% latex). In all gloves that passed the 1,000-ml test, herpes simplex virus type 1 was not recovered. Poliovirus was recovered infrequently (1.4% vinyl, 1.5% latex). Preliminary analyses suggest that the 1,000-ml water test has significantly increased sensitivity over the 300-ml water test in the detection of small holes in both vinyl and latex gloves that may allow the passage of viral particles. Gloves that pass a 1,000-ml water challenge are unlikely to allow the passage of a small virus such as poliovirus. Given that human immunodeficiency virus, hepatitis B virus and herpes simplex virus type 1 are larger particles than poliovirus, gloves that pass the 1,000-ml water test theoretically could provide better protection.     

Latex and vinyl nonsterile examination gloves: status report on laboratory evaluation of defects by physical and biological methods

We have reported previously (H. R. Kotilainen, J. P. Brinker, J. L. Avato, and N. M. Gantz, Arch. Intern. Med. 149:2749-2753, 1989) that the quality of nonsterile examination gloves available for clinical use may be extremely variable. In view of the concern over human immunodeficiency virus and hepatitis B virus transmission to health care workers, the continuing variability of gloves available for use, and the need for a simple and safe test, we have evaluated 2,500 vinyl (five brands) and 2,000 latex (four brands) gloves by the 300-ml and the newly proposed 1,000-ml water tests and for permeability to herpes simplex virus type 1 and poliovirus type 1, respectively. While all 300-ml watertight gloves were unlikely to leak herpes simplex virus type 1 (1.3% vinyl; 0.5% latex), poliovirus was recovered much more frequently (8.9% vinyl, 6.1% latex). In all gloves that passed the 1,000-ml test, herpes simplex virus type 1 was not recovered. Poliovirus was recovered infrequently (1.4% vinyl, 1.5% latex). Preliminary analyses suggest that the 1,000-ml water test has significantly increased sensitivity over the 300-ml water test in the detection of small holes in both vinyl and latex gloves that may allow the passage of viral particles. Gloves that pass a 1,000-ml water challenge are unlikely to allow the passage of a small virus such as poliovirus. Given that human immunodeficiency virus, hepatitis B virus and herpes simplex virus type 1 are larger particles than poliovirus, gloves that pass the 1,000-ml water test theoretically could provide better protection.     

Quantification of the emetic toxin cereulide in food products by liquid chromatography-mass spectrometry using synthetic cereulide as a standard

Bacillus cereus produces the emetic toxin cereulide, a cyclic dodecadepsipeptide that can act as a K(+) ionophore, dissipating the transmembrane potential in mitochondria of eukaryotic cells. Because pure cereulide has not been commercially available, cereulide content in food samples has been expressed in valinomycin equivalents, a highly similar cyclic potassium ionophore that is commercially available. This research tested the biological activity of synthetic cereulide and validated its use as a standard in the quantification of cereulide contents in food samples. The synthesis route consists of 10 steps that result in a high yield of synthetic cereulide that showed biological activity in the HEp-2 cell assay and the boar sperm motility assay. The activity is different in both methods, which may be attributed to differences in K(+) content of the test media used. Using cereulide or valinomycin as a standard to quantify cereulide based on liquid chromatography-mass spectrometry (LC-MS), the concentration determined with cereulide as a standard was on average 89.9% of the concentration determined using valinomycin as a standard. The recovery experiments using cereulide-spiked food products and acetonitrile as extraction solute showed that the LC-MS method with cereulide as a standard is a reliable and accurate method to quantify cereulide in food, because the recovery rate was close to 100% over a wide concentration range.     

Development and evaluation of an enzyme-linked immunosorbent assay with recombinant SAG2 for diagnosis of Toxoplasma gondii infection in cats

Cats are pivotal in the transmission of Toxoplasma gondii. To develop a sensitive and specific serodiagnostic method for feline toxoplasmosis, surface antigen 2 (SAG2) of T. gondii was expressed in Escherichia coli and its diagnostic potential evaluated in an enzyme-linked immunosorbent assay (ELISA). The ELISA with recombinant SAG2 (rSAG2) was able to differentiate very clearly between sera from cats experimentally infected with T. gondii and sera from normal cats. Serum samples collected from domestic cats in Japan were investigated by the ELISA, and the results were compared with those of a commercially available latex agglutination test (LAT) kit. Of the 192 samples screened, 42 (21.9%) were positive by ELISA. Among the 42 ELISA-positive samples, 39 were positive by LAT. There was a significant correlation between ELISA and LAT titers. All the 150 ELISA-negative samples were negative by LAT. These results indicate that the ELISA with rSAG2 expressed in E. coli should be a useful method for detection of T. gondii infection in cats.     

The Salmonella typhimurium/mammalian microsomal assay. A report of the U.S. Environmental Protection Agency Gene-Tox Program

The Salmonella assay has been in use for almost 15 years and can be defined as a routine test for mutagenicity and for predicting potential carcinogenicity. It detects the majority of animal carcinogens and consequently plays an important role in safety assessment. The test is also routinely used as the frontline screen for environmental samples (complex mixtures) isolated from air, water and food. This role will continue to remain an area of growth as or because sample volumes associated with these testing areas are generally very limited and more extensive testing is generally impossible. While this test, like all others, has some limitations, it is recommended that it be regularly included in all genetic testing batteries.     

Condom Negotiation,HIV Testing,and HIV Risks among Women from Alcohol Serving Venues in Cape Town,South Africa

Background

Women in South Africa are at particularly high-risk for HIV infection and are dependent on their male partners'' use of condoms for sexual risk reduction. However, many women are afraid to discuss condoms with male partners, placing them at higher risk of HIV infection.

Purpose

To examine the association between fear of condom negotiation with HIV testing and transmission risk behaviors, including alcohol use and sexual risks among South African women.

Method

Women (N = 1333) residing in a primarily Xhosa-speaking African township in Cape Town and attending informal alcohol-serving venues (shebeens) completed anonymous surveys. Logistic regression was used to test the hypothesis that fear of condom negotiation would be associated with increased risk for HIV.

Results

Compared to women who did not fear condom negotiation, those who did were significantly less likely to have been tested for HIV, were more likely to have experienced relationship abuse, and to report more alcohol use and more unprotected sex.

Conclusions

For women in South Africa, fear of condom negotiation is related to higher risk of HIV. HIV prevention efforts, including targeted HIV counseling and testing, must directly address gender issues.     

Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 6.7 for assessment of carcinogenic potential of chemicals

The Syrian hamster embryo (SHE) cell transformation assay (CTA) is an important in vitro method that is highly predictive of rodent carcinogenicity. It is a key method for reducing animal usage for carcinogenicity prediction. The SHE assay has been used for many years primarily to investigate and identify potential rodent carcinogens thereby reducing the number of 2-year bioassays performed in rodents. As for other assays with a long history of use, the SHE CTA has not undergone formal validation. To address this, the European Centre for the Validation of Alternative Methods (ECVAM) coordinated a prevalidation study. The aim of this study was to evaluate the within-laboratory reproducibility, test method transferability, and between-laboratory reproducibility and to develop a standardised state-of-the-art protocol for the SHE CTA at pH 6.7. Formal ECVAM principles for criteria on reproducibility (including the within-laboratory reproducibility, the transferability and the between-laboratories reproducibility) were applied. In addition to the assessment of reproducibility, this study helped define a standard protocol for use in developing an Organisation for Economic Co-operation and Development (OECD) test guideline for the SHE CTA. Six compounds were evaluated in this study: benzo(a)pyrene, 3-methylcholanthrene, o-toluidine HCl, 2,4-diaminotoluene, phthalic anhydride and anthracene. Results of this study demonstrate that a protocol is available that is transferable between laboratories, and that the SHE CTA at pH 6.7 is reproducible within- and between-laboratories.     

The preparation of an IgG diagnostic agent based on stained polyacrolein latexes for use in the latex agglutination reaction

IgG diagnosticum for measuring the concentration of 131I-labeled IgG antibodies to enteric antigen beta 1MA by the latex agglutination inhibition (LAI) test has been prepared on the basis of polyacrolein latexes. A method for the titration of anti-IgG antibodies with the use of the above diagnosticum has been developed, based on the late, agglutination (LA) test. The optimum conditions for the microtitration variant of the LA and LAI tests have been defined. High sensitivity, specificity and simplicity of analysis with the use of latex IgG diagnosticum have been demonstrated. The newly developed methods have been successfully used in laboratory trials of a new diagnostic radiopharmaceuticals for the assay of 131I-labeled antibodies in this preparation and for the detection of side effects of immunization on the recipients.     

An extraction free modified o-phthalaldehyde assay for quantifying residual protein and microbial biofilms on surfaces

Abstract

Biological contamination of surfaces in industry and healthcare is an important vector of disease transmission. Current assays for detecting surface-adherent contamination require extraction of biological soil. However, physical inaccessibility or poor solubility may limit recovery. Here, how the o-phthalaldehyde (OPA) protein assay can be modified to measure residual protein (modeled with bovine serum albumin) or biofilm on a surface without extraction is described. The assay limit of detection (LOD) for protein was 1.6 µg cm^?2. The detection threshold for Staphylococcus epidermis biofilm was 117 µg cm^?2. The clinical utility of the method was demonstrated for measurements taken from clinically used endoscopes. Since this method is more sensitive than extraction-based testing, clinical results should not be compared with conventional benchmarks. By enabling direct detection and quantification of soils in complex or hard-to-reach areas, this method has potential to improve the margin of safety in medical and industrial cleaning processes.     

Reproductive biology of invertebrates. Volumes I and II: Edited by K. G. Adiyodi and R. G. Adiyodi,Wiley, New York, 1983. 770 pp. and 692 pp. $110.00 and $94.00

Biological control agents (biorationals) are increasingly important in pest control concepts. Certain insect viruses, particularly the baculoviruses (nuclear polyhedrosis viruses), are considered to have potential as biological pesticides and could be used widely in the environment. Therefore, test animals must be selected and methods and laboratory systemsdeveloped to evaluate the safety of these agents to nontarget species. A simple laboratory system has been designed and used to determine risks of infectivity and pathogenicity of an insect Baculovirus, originally isolated from the Alfalfa looper, Autographa californica, to a nontarget arthropod, the grass shrimp, Palaemonetes vulgaris, by dietary exposure. This laboratory method also permits evaluation of other microbial biorationals against nontarget aquatic species, and provides an inexpensive standardized procedure of safety testing. Results from this study indicated that histopathological, ultrastructural, and serological methods used provided no evidence that experimental exposure to the virus in our test system caused viral infection or related pathogenicity in the grass shrimp.     

International Society for Analytical Cytology biosafety standard for sorting of unfixed cells.

BACKGROUND: Cell sorting of viable biological specimens has become very prevalent in laboratories involved in basic and clinical research. As these samples can contain infectious agents, precautions to protect instrument operators and the environment from hazards arising from the use of sorters are paramount. To this end the International Society of Analytical Cytology (ISAC) took a lead in establishing biosafety guidelines for sorting of unfixed cells (Schmid et al., Cytometry 1997;28:99-117). During the time period these recommendations have been available, they have become recognized worldwide as the standard practices and safety precautions for laboratories performing viable cell sorting experiments. However, the field of cytometry has progressed since 1997, and the document requires an update. METHODS: Initially, suggestions about the document format and content were discussed among members of the ISAC Biosafety Committee and were incorporated into a draft version that was sent to all committee members for review. Comments were collected, carefully considered, and incorporated as appropriate into a draft document that was posted on the ISAC web site to invite comments from the flow cytometry community at large. The revised document was then submitted to ISAC Council for review. Simultaneously, further comments were sought from newly-appointed ISAC Biosafety committee members. RESULTS: This safety standard for performing viable cell sorting experiments was recently generated. The document contains background information on the biohazard potential of sorting and the hazard classification of infectious agents as well as recommendations on (1) sample handling, (2) operator training and personal protection, (3) laboratory design, (4) cell sorter set-up, maintenance, and decontamination, and (5) testing the instrument for the efficiency of aerosol containment. CONCLUSIONS: This standard constitutes an updated and expanded revision of the 1997 biosafety guideline document. It is intended to provide laboratories involved in cell sorting with safety practices that take into account the enhanced hazard potential of high-speed sorting. Most importantly, it states that droplet-based sorting of infectious or hazardous biological material requires a higher level of containment than the one recommended for the risk group classification of the pathogen. The document also provides information on safety features of novel instrumentation, new options for personal protective equipment, and recently developed methods for testing the efficiency of aerosol containment.     

Comparison of in vitro assays of cellular toxicity in the human hepatic cell line HepG2

Cytotoxicity testing allows determining whether a compound or extract contains significant quantities of biologically harmful chemicals. Cytotoxicity test methods are useful for screening because they serve to separate toxic from nontoxic materials, providing predictive evidence of compound safety. However, a wide range of assays measuring different aspects of cell death is available in the market, but it is difficult to determine which one(s) to use when evaluating a selection of compounds. The objective of this study was to compare different commercially available in vitro assays for cytotoxicity in HepG2 cells according to its sensitivity, reproducibility, simplicity, cost, and speed. The assays evaluated included Alamar Blue for the measurement of mitochondrial activity, ATPlite and ViaLight for the determination of cellular adenosine triphosphate (ATP), ToxiLight as an indicator of cellular necrosis, and Caspase-3 Fluorometric Assay, Apo-ONE Caspase-3/7 Homogeneous Assay, and Caspase-Glo for the determination of caspase-3/7 activity. All assays were performed using 4 compounds of previously reported cytotoxic activity: DMSO, butyric acid, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and camptothecine. Overall, it was concluded that the best way to evaluate the potential cytotoxicity of a compound is to employ a battery of assays that focus on different aspects of cell death. In this case, the focus has been on ATP levels, cell necrosis, and capsase-3/7 activation. Many other kits are commercially available in the market for these and other aspects of necrosis and/or apoptosis. However, the use of ViaLight Plus, ToxiLight, and Caspase-3 Fluorometric Assay resulted in the most useful combination when working with HepG2 cells.     

Development of a semi-high-throughput growth assay for the filamentous actinobacteria <Emphasis Type="Italic">Frankia</Emphasis>

Filamentous bacteria pose unique challenges for testing multiple variables or growth parameters limiting the use of high-throughput methods. A semi-high-throughput growth assay system was developed to overcome these obstacles and validated for the filamentous actinobacteria Frankia. The 24-well plate assay was versatile for testing multiple growth medium parameters and provided reproducible results across wells and between plates. Under conditions of increased complexity, statistical analysis demonstrated that the variance was dependent on the experimental parameters and not the assay system. The 24-well plate assay was shown to be multipurpose for testing numerous variables on cell growth or other biological properties.     

A radioreceptor assay for pharmaceutical preparations of insulin

A radioreceptor assay has been developed that is suitable for the measurement of the potency of crystalline insulin and pharmaceutical insulin formulations. It utilizes the well characterized and widely available IM-9 human lymphocyte cell line as the source of receptor. Bovine, porcine and human crystalline and formulated insulins have been assayed against the 4th International and European Standards for Insulin and the potencies compared with those obtained by the mouse blood glucose method. Results with bovine insulin were in full correspondence with the in vivo results. Porcine and human insulins were 15-20% more potent by the radioreceptor assay than by the in vivo method when the mixed bovine and porcine insulin 4th International and European Standards were used, but were equivalent when compared with like materials. Average 95% confidence limits for formulated insulins in two assays were +/- 6% of the mean. The coefficient of variation on repeated assay of the same sample was 3.8%. The three dose parallel line radioreceptor assay with appropriate species species standards is a candidate biological test capable of international adoption as an alternative to in vivo animal testing of insulin.     

A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite.

Numerous methods are available for measurement of nitrate (NO(-)(3)). However, these assays can either be time consuming or require specialized equipment (e.g., nitrate reductase, chemiluminescent detector). We have developed a method for simultaneous evaluation of nitrate and nitrite concentrations in a microtiter plate format. The principle of this assay is reduction of nitrate by vanadium(III) combined with detection by the acidic Griess reaction. This assay is sensitive to 0.5 microM NO(-)(3) and is useful in a variety of fluids including cell culture media, serum, and plasma. S-Nitrosothiols and L-arginine derivatives were found to be potential interfering agents. However, these compounds are generally minor constituents of biological fluids relative to the concentration of nitrate/nitrite. This report introduces a new, convenient assay for the stable oxidation products of nitrogen oxide chemistry in biological samples.     

A latex agglutination assay for specific detection of Panton-Valentine leukocidin

Panton-Valentine leukocidin (PVL) is produced by some isolates of Staphylococcus aureus, and has been associated with the high pathogenic potential of these strains. To rapidly detect the toxin producer strains, we developed a reverse passive latex agglutination (RPLA) reaction assay specific for PVL. By testing 64 S. aureus strains, the assay could detect the 35 pvl-gene-positive strains with 100% specificity and sensitivity. Furthermore, the assay revealed an extensive variation in the amount of PVL produced by the pvl-positive strains.