Analysis of results from a collaborative study of the dominant lethal assay

A dominant lethal (DL) mutation is a genetic change that results in the death of the conceptus that inherits it. The assya is normally carried out in mice and the production of DL mutation by the test chemical is characterised by an increase in non-viable embryos.A distinct advantage of the DL assay is that it is an in vivo, mammalian, germ cell assay and is therefore pertinent to the fundamental question of chemical mutagenesis, i.e.: is a chemical likely to produce genetic changes that can be transferred to the offspring? The two principal criticisms of the assay are that the main type of genetic damage it detects is chromosomal breakage that leads to the conceptus, and that the assay is considered to be relatively insensitive.The Association of the British Pharmaceutical Industry (ABPI) Working Party on Mutagenicity was established in 1974. Its principal objective was to consider the problem of mutagenicity and associated matters in relation to the development of new medicines and to make recommendations and proposals for collaborative work if indicated. The dominant lethal assay is one of several tests for mutagenicity chosen for evaluation by collaborative study. The objective of the collaborative study was to gain more information about the validity and dependability of the dominant lethal assay.The present report concerns an analysis of the differences between compounds, laboratories and statistical methods utilising the data from the collaborative study.     

Avoiding false negative results in specificity analysis of protein-protein interactions

The competition measurement using simultaneous incubation of labeled and unlabeled Ligand is a common method to assess the specificity of a biomolecular interaction. In this paper we show that invalid assumptions about the interactions may lead to improper experimental setups which in turn can result in inaccurate conclusions about the specificity. To improve understanding of competition measurements, simulations in MATLAB as well as real-time interaction analysis using LigandTracer have been performed. We show that use of a concentration of unlabeled Ligand of at least 10 × K(D) is necessary for assay accuracy. Increasing the incubation time to assure equilibrium, adding a pre-incubation phase, and a general understanding of the reversibility of an interaction may also improve the reliability of the measurement and the conclusions drawn about specificity. These findings may lower the risk of false negative results as well as reducing the amount of reagent needed.     

Psychological consequences for parents of false negative results on prenatal screening for Down's syndrome: retrospective interview study

ObjectiveTo determine the psychological consequences for parents of children with Down''s syndrome of having received a false negative result on prenatal screening.DesignComparison of adjustment of parents who received a false negative result with that of parents not offered a test and those who declined a test.SettingParents were interviewed in their own homes.ParticipantsParents of 179 children with Down''s syndrome (mean age 4 (range 2-6) years).ResultsOverall, regardless of screening history, parents adjusted well to having a child with Down''s syndrome. Compared with mothers who declined a test, mothers in the false negative group had higher parenting stress (mean score 81.2 v 71.8, P=0.016, 95% confidence interval for the difference 1.8 to 17.0) and more negative attitudes towards their children (124.9 v 134.2, P=0.009, −16.2 to −2.4). Fathers in the false negative group had higher parenting stress test scores (77.8 v 70.0, P=0.046, 1.5 to 14.2) than fathers not offered a test. Mothers in the false negative group were more likely to blame others for the outcome than mothers who had not been offered the test (28% v 13%, P=0.032, 3% to 27%). Mothers and fathers in the false negative group were more likely to blame others for this outcome than parents who had declined a test (mothers 28% v 0%, P=0.001, 19% to 37%; fathers 27% v 0%, P=0.004, 17% to 38%). Blaming others was associated with poorer adjustment for mothers and fathers.ConclusionsA false negative result on prenatal screening seems to have a small adverse effect on parental adjustment evident two to six years after the birth of an affected child.     

An international collaborative study on foot and mouth disease virus assay methods. 1. Virus infectivity and neutralizing antibody assays

In a collaborative study sponsored by the European Commission for the Control of Foot and Mouth Disease, workers in 19 laboratories participated in the early phases (1, 2 and 4). All three phases were devoted to assessments of virus infectivity and the neutralizing activity of sera. Virus preparations and antisera distributed from one laboratory were tested either by 'in house' or suggested methods. Analyses of the results clearly showed that whilst 'within' laboratory variation in the results of replicate tests was low, there were significant differences in the results from various laboratories. By attempting to standardize test conditions with distributed materials, e.g. media and cells, etc., the 'between' laboratory variation was reduced.     

Avoiding false positive HIV results on life assurance proposers: the necessity for follow-up

The serological reaction to HIV infection is almost invariably a dynamic progression towards strong reactivity to a wide range of viral antigens. Serological diagnosis should therefore be based either on the presence of this wide range in a single specimen or on the demonstration of increasing activity between two specimens collected at an interval of two or more weeks and tested in parallel. When a specimen is reactive a second should in any case be collected to check the identity of the first. These precepts are sometimes being ignored, especially in non-clinical testing, e.g., for purpose of life assurance. Two recent cases in which there were false but unchanging enzyme immunoassay and Western blot reactions that might have been interpreted as positive illustrate the potential for error.     

Estimating false positive and false negative error rates in cervical cell classification.

The performance of a cell recognition system on unknown data is often estimated in terms of its error rates on a test set. This paper investigates methods for producing estimates of error rates in cervical cell classification. Classification performance curves calculated using these methods are given for several classification schemes used to classify 1500 cervical cells.     

A European collaborative study of the Ames assay: I. Results and general interpretation

Results are presented of a collaborative study between 19 European laboratories on the variability of the Ames test. Examples are shown of various methods that are generally used to evaluate an Ames test without reference to a specific statistical model: the number of revertants per plate, mutation factors (increase over the spontaneous value) and determination of the doubling concentration.Considerable variations between test results occurred, between laboratories as well as within laboratories. Partly this was due to different interpretations of the guidelines given, as these allowed some flexibility. The results were also influenced by other factors, some of which are perhaps not yet generally recognized. Apart from the level and quality of the S9 preparations, the most important factor might be the number and physiological condition of the cells plated.When the results from all experiments were considered together, 60–80% of the test results were found within the half- to two-fold range of the overall median. This might be considered satisfactory for a study not using rigidly standardized test conditions.From the experience with the present study, several recommendations are given for the design and performance of future collaborative studies.     

Impact of new antiretroviral combination therapies in HIV infected patients in Switzerland: prospective multicentre study. Swiss HIV Cohort Study.

OBJECTIVES: To examine trends in disease progression and survival among patients enrolled in the Swiss HIV cohort study during 1988-96 and to assess the influence of new antiretroviral combination therapies. DESIGN: Prospective multicentre study, with follow up visits planned at six monthly intervals. SETTING: Seven HIV units at university centres and cantonal hospitals in Switzerland. PATIENTS: 3785 men (mean age 35.0 years) and 1391 women (30.3 years) infected with HIV. 2023 participants had a history of intravenous drug misuse; 1764 were men who had sex with men; 1261 were infected heterosexually; and 164 had other or unknown modes of transmission. 601 participants had had an AIDS defining illness. RESULTS: During more than 15,000 years of follow up, there were 1456 first AIDS defining diagnoses and 1903 deaths. Compared with those enrolled during 1988-90, the risk of progression to a first AIDS diagnosis was reduced by 18% (relative risk 0.82 (95% confidence interval 0.73 to 0.93)) among participants enrolled in 1991-2, by 23% (0.77 (0.65 to 0.91)) among those enrolled in 1993-4, and by 73% (0.27 (0.18 to 0.39)) among those enrolled in 1995-6. Mortality was reduced by 19% (0.81 (0.73 to 0.90)), 26% (0.74 (0.63 to 0.87)), and 62% (0.38 (0.25 to 0.97)) respectively. Compared with no antiretroviral treatment, the risk of an initial AIDS diagnosis after CD4 lymphocyte counts fell to < 200 cells x 10(6)/1 was reduced by 16% (0.84 (0.73 to 0.97)) with monotherapy, 24% (0.76 (0.63 to 0.91)) with dual therapy, and 42% (0.58 (0.37 to 0.92)) with triple therapy. Mortality was reduced by 23% (0.77 (0.68 to 0.88)), 31% (0.69 (0.60 to 0.80)), and 65% (0.35 (0.20 to 0.60)) respectively. CONCLUSIONS: The introduction of antiretroviral combination therapies outside the selected patient groups included in clinical trials has led to comparable reductions in disease progression and mortality.     

Risk of HIV infection from transfusion with blood negative for HIV antibody in a west African city.

OBJECTIVE--To estimate the risk of infection with HIV (HIV 1 or HIV 2, or both) from transfusion of a screened unit of blood in a high prevalence area in west Africa. DESIGN--Retrospective cohort study for January-July 1991. SETTING--National Blood Transfusion Centre, Abidjan, Côte d''Ivoire. SUBJECTS--Repeat donors (5831 units of blood) and first time donors (5076 units) in the first five months of 1991. MAIN OUTCOME MEASURES--Prevalence and estimated incidence of HIV infection in repeat and first time donors; estimated rate of potentially infected, HIV antibody negative units; and rate of (false negative) potentially infected units assuming a laboratory test sensitivity of 99%. RESULTS--Overall HIV prevalence was 11.0% in first time donors and 2.1% in repeat donors. In the first seven months of 1991, 29 HIV antibody positive (27 HIV 1, 1 HIV 2, 1 dually reactive) donors with a seronegative unit of blood earlier in the year were identified; 26 had donated blood eight weeks or less before their estimated dates of seroconversion and may have been infectious (minimum rate 26/5831 (4.5/1000 potentially infected units)). Estimated incidence of infection in repeat donors was 1.2-2.5%. Laboratory test insensitivity would result in an estimated 1.1/1000 false negative units from first time donors and 0.2/1000 units from regular donors. The overall rate of potentially infected units (all donors, seroconversions, and errors) was estimated at 5.4-10.6/1000. CONCLUSIONS--The risk of HIV infection from a single unit of blood remains substantial (5.4-10.6/1000 units). To prevent infection from blood transfusion in areas of high incidence and prevalence of HIV all but absolutely essential transfusions should be avoided, and donors with low incidence of HIV infection should be selected.     

Microplate biochemical determination of Russian VX: influence of admixtures and avoidance of false negative results

Two microplate spectroscopic methods for determination of organophosphates, based on inhibition of acetylcholinesterase (AChE) activity, were further improved and evaluated for determination of the chemical weapon agent Russian VX (RVX) in aqueous solutions. The linear range of the Hestrin method (74.8-1120 pM) was 3.1-fold wider than that of the Ellman method (37.4-374 pM). Limits of detection and quantification of RVX for both methods were below the maximal allowable concentration of RVX in water-soluble washouts. One of the early products of RVX hydrolysis, N,N-diethylaminoethanethiol, like reduced glutathione, caused false negative results in the Ellman method at concentrations exceeding 10 μM; individual blanks were necessary to eliminate the effect. The Hestrin method showed greater specificity (~3 orders of magnitude) for analysis of samples containing mercaptans. A major product of RVX degradation, 2,2'-dithiobis(N,N-diethylethanamine), caused significant inhibition of AChE at concentrations of ≥0.1 mM (P<0.01) and had a false positive effect at higher concentrations (≥2 mM). For environmental monitoring of RVX, the method based on Hestrin is preferred over that based on Ellman, principally because the former method was less sensitive to interference from major admixtures and did not give rise to potentially dangerous false negative results.     

Ethylene dibromide: negative results with the mouse dominant lethal assay and the electrophoretic specific-locus test.

Ethylene dibromide (1,2-dibromoethane; EDB) was tested for the induction of dominant lethal and electrophoretically-detectable specific-locus mutations in the germ cells of DBA/2J male mice. Males were treated with a single intraperitoneal injection of 100 mg/kg EDB and mated to two C57BL/6J females. In the dominant lethal assay, matings were carried out to measure the effect of EDB on meiotic and postmeiotic stages; germ cells representing spermatogonial stem cells were analyzed in the electrophoretic specific-locus test. Neither of these germ cell tests produced any evidence that EDB is a germ cell mutagen. It appears from these data and those reported in the literature that EDB, a genotoxic carcinogen that affects male fertility in some mammalian species, is not mutagenic in the germ cells of the male mouse.     

Potency assay of antibodies against rabies. A report on a collaborative study

The relative potencies of a number of rabies immunoglobulin preparations were estimated in an international collaborative study comprising eight laboratories in five countries. Two assay methods were used: a virus neutralization test in mice (MNT) and a virus neutralization test in cell culture (RFFIT). Differences between the results obtained by the two methods, which have been reported, could not be generally corroborated. The results indicate that in some laboratories the MNT cause difficulties and give results different from those obtained by RFFIT. In other laboratories such difficulties are not encountered. The results seem to indicate that the RFFIT is a more reliable method than the MNT.     

The reasons for false positive results with recombinant preparations in HIV infection]

Enzyme immunoassay (EIA) with the use of test systems based on recombinant HIV antigens has yielded positive results much more often than that with the test systems based on HIV lysate. In comparison with the use of protein A-peroxidase conjugate, the use of antiglobulin conjugates decreased the specificity of EIA, thus ruling out the false positive results. The tested sera containing antibodies to antigens of enteric bacilli could yield false positive EIA results with test systems prepared from recombinant antigens due to the interaction of these antibodies with antigens of enteric bacilli. A conclusion on the usefulness of the control for the presence of the components of enteric bacilli, producers of HIV antigen, and their elimination from recombinant preparations is made.     

Cellularity of lobular carcinoma and its relationship to false negative fine needle aspiration results

OBJECTIVE: To evaluate the cytocellularity and histocellularity of lobular carcinoma (LC) and the relationship to high false negative results of fine needle aspiration cytology (FNAC). STUDY DESIGN: In this retrospective study, cellularity was studied in 60 cases of classic LC, LC variants and lobular carcinoma in situ, comparing cytologic smears to their corresponding histologic sections. The cytologic smears were grouped into acellular, low, moderate and high categories, and the histologic sections were grouped into low, moderate and high categories. RESULTS: Malignancy or suspicion of malignancy was diagnosed in 78% of cases. Overall cytocellularity showed acellularity or low cellularity in 60% of cases, while overall histocellularity showed moderate or high cellularity in 95% of cases. When the cytocellularity was moderate or high, the corresponding histocellularity always showed moderate or high histocellularity. When the cytocellularity was low, the corresponding histology showed low histocellularity in 6.3% of cases. Thus, in acellular and low cellular aspirations, corresponding histocellularity may not be the contributing factor toward low cellular yield. In this study, 22% of cases were diagnosed as false negative, 40% were diagnosed as suspicious, and 38% were called positive. Only 17% of positive cases were diagnosed as LC. A large number of LC were misdiagnosed by FNAC as duct cell carcinoma, and most cases of low histocellularity were of the classic type. CONCLUSION: The results of this study suggest that in the majority of cases of LC, cellular yield of FNAC is disproportionately lower than expected when compared to the corresponding histocellularity. Awareness of modest cellularity and subtle cytologic features will aid in the correct preoperative diagnosis of LC, and false negative diagnoses can be minimized.