The minimum significant ratio: a statistical parameter to characterize the reproducibility of potency estimates from concentration-response assays and estimation by replicate-experiment studies

The authors show by illustration that procedures used to validate the reliability of single-concentration high-throughput screens such as the signal window and Z' factor do not ensure sufficient reliability in potency estimates from concentration response assays. They develop the minimum significant ratio as a statistical parameter to characterize the fold change between 2 compounds run in the same experiment that can be considered a real difference and use this parameter to characterize the reliability of the assay. They adapt methods described by Bland and Altman to develop a simple set of 2 experiments to estimate the minimum significant ratio and show that this protocol can identify assays that lack reproducibility. The methods are then extended to validate the equivalency of the same assay run by multiple laboratories.     

Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.     

Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: II. With exogenous activation

In order to determine the usefulness of selected chemicals as potential reference materials for calibrating the Salmonella assay, two laboratories tested a series of Salmonella mutagens that require exogenous activation. When the variance for individual substances within a bioassay is sufficiently low and the rankings of those substances are of acceptable consistency, they can later be evaluated for use as standard control compounds, as audit materials, and as standard reference materials for comparative bioassay efforts. The purpose of this project, therefore, was to evaluate the variability in the mutagenic response of potential reference chemicals that require exogenous metabolic activation in the standard plate-incorporation Salmonella mutagenicity assay, and to develop ranking criteria for mutagenic activity based on these data. Ten indirect-acting mutagens were tested in two laboratories using Salmonella typhimurium TA100 and an Aroclor-induced rat liver S9. Each laboratory conducted four definitive testing rounds. A different batch of S9 was utilized for every two rounds. Of the 10 chemicals tested only 2-anthramine had a mean slope value greater than 1000 revertants/micrograms. Three chemicals had slope values between 1000 and 100; and five chemicals had slope values between 100 and 10. The remaining compound, 9,10-dimethyl-1,2-benz[a]anthracene, could not be placed into a single category because it had slope values on either side of 100 revertants per mg. Coefficients of variance were low (i.e., below 25% in most cases). The low variability achieved in this study may be accounted for by two parameters of the study. First, based on Claxton et al. (1991a) and the S9 optimization for three compounds, the amount of S9 was calibrated to a set amount of protein per plate (1.1 mg/plate). Secondly, the 10 test doses were placed in the initial, linear, nontoxic portion of the dose-response curves. The use of ten closely spaced, nontoxic doses allowed for a more accurate estimate of the slope.     

Tradescantia stamen hair mutation bioassay

The Tradescantia stamen hair mutation (Trad-SH) assay (clone 4430) was evaluated for its efficiency and reliability as a screen for mutagens in an IPCS collaborative study on plant systems. Four coded chemicals, i.e. azidoglycerol (AG, 3-azido-1,2-propanediol), N-methyl-N-nitrosourea (MNU), sodium azide (NaN₃) and maleic hydrazide (MH) were distributed by the Radian Corporation to the five laboratories in five different countries for testing mutagenicity. Pink mutations were scored between the 7th and 14th day according to a standard protocol. Test results from the five individual laboratories were analyzed and compared after decoding. One out of the two laboratories that conducted tests on AG demonstrated that AG is a mutagen with genetically effective doses ranging from 50 to 100 μg/ml. MH yielded positive responses in all laboratories but no linear dose-response pattern was observed. The effective dose range for MH was between 1 and 45 μg/ml. The mutagenicity of MNU was reported by five laboratories in the dose range between 10 and 80 μg/ml. NaN₃, which exhibited a relatively high degree of toxicity, elicited a positive mutagenic response in three of the five laboratories in which it was tested. As with MNU the effective dose for NaN₃ ranged between 3 and 80 μg/ml. The results from the current study substantiate the Trad-SH assay as a reliable system for screening chemicals for their potential mutagenic effects. Although the study was carried out exclusively under laboratory conditions, a survey of the current literature would indicate that the Trad-SH assay could be an effective in situ monitor of gaseous, liquid, and radioactive pollutants as well.     

The Pertussis Serological Potency Test. Collaborative study to evaluatereplacement of the Mouse Protection Test.

The Pertussis Serological Potency Test (PSPT)--based on in vitro assessment of the humoral immune response against Bordetella pertussis--was developed as an alternative for the Mouse Protection Test (MPT). A small-scale collaborative study was carried out in five laboratories to evaluate the relevance and reliability of the PSPT. The study has been divided into three separate phases, each with its own objective. A pilot-phase study of the antibody detection assay, the 18323-whole cell ELISA (WCE), was included for training purposes. Significant differences in absorbance and antibody concentrations between the laboratories were found. In the Phase I study, the intra-assay, inter-assay and inter-laboratory precisions of the 18323-WCE were assessed. Although a precision of less than 20% was not always established and significant differences in antibody concentrations were found at random throughout the Phase I study, the ranking of the antibody concentrations corresponded well between the laboratories and should warrant a reliable potency estimation of whole cell vaccines (WCV's) in the PSPT. Phase II was a comparative study of the PSPT and the MPT to evaluate the implementation of the PSPT, to demonstrate correlation and to compare the reproducibility and reliability of both tests. The mean antibody concentrations per vaccine dose in the PSPT and the survival of mice in the MPT differed significantly within and between the laboratories. Nevertheless, the potencies of the vaccines under test estimated in both test models did not differ significantly (P>0.05). The PSPT and MPT correlated well in chi2-test of homogeneity within and between the laboratories. The potencies were similar (overall ratio=0.877), but the PSPT is more reproducible and reduces the chance of re-testing due to the smaller 95% confidence intervals. We have demonstrated that the PSPT is a valid model to estimate the potencies of pertussis WCV's from different manufacturers. Moreover, the 18323-WCE is easy to carry out and the intra-assay precision and antibody ranking warrants a reliable potency testing of pertussis WCV's in the PSPT.     

A sensitive branched DNA HIV-1 signal amplification viral load assay with single day turnaround

Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) (“Versant Assay”) currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16–18 h to 2.5 h, composition of only the “Lysis Diluent” solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements.     

An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.     

External quality assurance of the carcinoembryonic antigen (CEA) assay: main findings in six years' experience.

In 1984 we initiated a national external quality assessment (EQA) program (supported by the Italian National Research Council, CNR) for the CEA assay; at present, about 200 Italian laboratories are participating in the program. The laboratories assayed the quality control (QC) samples according to their routine procedures and returned the results together with the name of the method/kit they used. The collected results were computer-processed and reports were sent back to the participants. A significant reduction of the CVt (mean between-laboratory agreement) of the CEA assay was observed throughout the EQA survey (from 35% in 1985 to 20-25% in the last cycles). In order to better clarify the differences in variability observed in the first QC cycles against the last ones, we used the ANOVA technique to evaluate the components of variability. The improvement in between-laboratory agreement was mainly due to the reduction of the between-kit component (from 30.5% to 15.2%), rather than to the smaller decrease observed for the within-kit variability (from 18.4% to 14.0%). The results reported for QC samples from different materials showed differences in the between-lab variability and substantial changes of the kit biases, thus suggesting a different specificity of the antibodies used in the various method/kits against different families of CEA molecules. Considerable uncertainty was also encountered in the clinical classification of low pathological samples, which seems mainly due to the variability in cut-off values used by the laboratories for the clinical assessment of the same analytical results. Our data indicate a progressive increase in the reliability of CEA determination during our study and confirm that EQA has improved the reliability of analysis carried out by the participating laboratories, thus stimulating the kit manufacturers to provide more reliable products.     

Adaptation of the hypoosmotic swelling test to assess functional integrity of stallion spermatozoal plasma membranes

Hypoosmotic swelling (HOS) is used for assessing plasma membrane function and fertilizing capacity of human spermatozoa. However, HOS solutions and methodologies have not been evaluated specifically for assessing stallion spermatozoa. The objective of this study was to identify a HOS solution and assay conditions specifically for stallions that would maximize spermatozoal plasma membrane swelling. The HOS solutions and assay conditions, including incubation time (15 to 180 min), temperature (25 degrees vs 37 degrees C), and total number of cells examined (100, 200 or 500) were evaluated. Assay consistency, accuracy, reliability and repeatability also were determined. Maximum spermatozoal plasma membrane swelling was observed in a 100 mosmol sucrose solution (P < 0.001). Incubation time (P = 0.67), temperature (P = 0.70) and total number of spermatozoa examined (P = 0.38 and P = 0.24 for 100 vs 200 and 100 vs 500, respectively) did not influence percent of HOS positive spermatozoa observed. A high degree of assay accuracy was indicated when a correlation of r = 0.998 was obtained between the HOS positive spermatozoa observed and expected when known amounts of heat-treated spermatozoa, unable to undergo swelling, were added to untreated spermatozoa. Assay consistency was demonstrated, as reflected by a mean coefficient of variation of 0.073 for 4 stallions. Also, the coefficient of variation from 2 analyses of variance was 0.168 and 0.096, indicating reasonably good assay reliability; estimates of repeatability from the same analyses were 0.794 and 0.968. The HOS test adapted to stallion spermatozoa in this study is a simple, highly accurate and consistent assay with good reliability and repeatability. Results observed under the conditions evaluated also permit some flexibility in adapting this assay to individual laboratory and practice settings for evaluating stallion spermatozoal plasma membranes.     

Statistical diagnostics emerging from external quality control of real-time PCR

Besides the application of conventional qualitative PCR as a valuable tool to enrich or identify specific sequences of nucleic acids, a new revolutionary technique for quantitative PCR determination has been introduced recently. It is based on real-time detection of PCR products revealed as a homogeneous accumulating signal generated by specific dyes. However, as far as we know, the influence of the variability of this technique on the reliability of the quantitative assay has not been thoroughly investigated. A national program of external quality assurance (EQA) for real-time PCR determination involving 42 Italian laboratories has been developed to assess the analytical performance of real-time PCR procedures. Participants were asked to perform a conventional experiment based on the use of an external reference curve (standard curve) for real-time detection of three cDNA samples with different concentrations of a specific target. In this paper the main analytical features of the standard curve have been investigated in an attempt to produce statistical diagnostics emerging from external quality control. Specific control charts were drawn to help biochemists take technical decisions aimed at improving the performance of their laboratories. Overall, our results indicated a subset of seven laboratories whose performance appeared to be markedly outside the limits for at least one of the standard curve features investigated. Our findings suggest the usefulness of the approach presented here for monitoring the heterogeneity of results produced by different laboratories and for selecting those laboratories that need technical advice on their performance.     

Critical appraisal of the side population assay in stem cell and cancer stem cell research

The "Side Population" (SP) discrimination assay is a flow cytometry method used to detect stem cells based on the dye efflux properties of ABC transporters. We discuss the SP assay and its applications in stem cell?biology, with an emphasis on the technical challenges related to sample preparation, data acquisition, analysis, and interpretation. We highlight the value of multicolor phenotyping, the impact of DNA ploidy, and the importance of distinguishing graft versus host cells for an appropriate SP discrimination. To improve the consistency and reliability of data between laboratories, we propose a set of recommendations for SP assay data reporting.     

Vicia faba chromosomal aberration assay

A collaborative study involving laboratories in six countries was initiated under the sponsorship of the International Programme on Chemical Safety (IPCS) to determine the sensitivity, efficiency and reliability of the Vicia faba root tip meristem chromosomal aberration assay using a standardized protocol. The six Laboratories that participated in this study were located in the Slovak Republic, India, Japan, Poland, Sweden and the USA. All laboratories adhered to a standardized protocol for the Vicia faba chromosomal aberartion assay. Four coded chemicals, azidoglycerol (AG), N-methyl-N-nitrosourea (MNU), sodium azide (NaN₃) and maleic hydrazide (MH) were tested with the Vicia faba chromosomal aberration assay. Of the four chemicals, three (MH, AG and MNU) were found to be clastogenic and gave a concentration related response. However, the results of NaN₃ were equivocal which might be explained by the stability of NaN₃. The conclusions from this study suggest that the Vicia faba chromosomal aberration bioassay is an efficient and reliable short-term bioassay for the rapid screening of chemicals for clastogenicity.     

Effects of assay conditions in life history experiments with Drosophila melanogaster

Selection experiments with Drosophila have revealed constraints on the simultaneous evolution of life history traits. However, the responses to selection reported by different research groups have not been consistent. Two possible reasons for these inconsistencies are (i) that different groups used different environments for their experiments and (ii) that the selection environments were not identical to the assay environments in which the life history traits were measured. We tested for the effect of the assay environment in life history experiments by measuring a set of Drosophila selection lines in laboratories working on life history evolution with Drosophila in Basel, Groningen, Irvine and London. The lines measured came from selection experiments from each of these laboratories. In each assay environment, we measured fecundity, longevity, development time and body size. The results show that fecundity measurements were particularly sensitive to the assay environment. Differences between assay and selection environment in the same laboratory or differences between assay environments between laboratories could have contributed to the differences in the published results. The other traits measured were less sensitive to the assay environment. However, for all traits there were cases where the measurements in one laboratory suggested that selection had an effect on the trait, whereas in other laboratories no such conclusion would have been drawn. Moreover, we provide good evidence for local adaptation in early fecundity for lines from two laboratories.     

Interlaboratory assessment of the GreenScreen HC GADD45a-GFP genotoxicity screening assay: an enabling study for independent validation as an alternative method

Sixteen coded compounds were blind-tested at 4 laboratories using the recently described GADD45a-GFP genotoxicity assay. The compounds were chosen to include non-genotoxic compounds as well as weak and strong genotoxins. None of the compounds required metabolic activation in order to exhibit genotoxic effects. The participating laboratories included 2 global pharmaceutical companies, a global consumer goods company and the Gentronix laboratory in Manchester. Each compound was tested 4 times on different days following a protocol previously described. The tests were carried out after a 3-day training period from the parent lab (Manchester). Following the exclusion of data from tests with positive control failures and data series with 'spikes', 92% of assays gave the correct result: non-genotoxins giving negative results and genotoxins giving positive results. There were no randomly distributed problems suggesting that differences between the results from different sites reflected the use of different instruments, procedural differences and operator experience. In na?ve operator laboratories the quality of data improved with operator practice. It was concluded that simple clarification of the protocol would provide the level of reliability required for widespread use of the assay in hazard assessment.     

Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.     

CD45 function is regulated by an acidic 19-amino acid insert in domain II that serves as a binding and phosphoacceptor site for casein kinase 2

In this study experiments were conducted to elucidate the physical/functional relationship between CD45 and casein kinase 2 (CK2). Immunoprecipitation experiments demonstrated that CK2 associates with CD45 and that this interaction is inducible upon Ag receptor cross-linking in B and T cell lines as well as murine thymocytes and splenic B cells. However, yeast two-hybrid analysis failed to demonstrate a physical interaction between the individual CK2 alpha, alpha', or beta subunits and CD45. In contrast, a yeast three-hybrid assay in which either CK2 alpha and beta or alpha' and beta subunits were coexpressed with the cytoplasmic domain of CD45, demonstrated that both CK2 subunits are necessary for the interaction with CD45. Experiments using the yeast three-hybrid assay also revealed that a 19-aa acidic insert in domain II of CD45 mediates the physical interaction between CK2 and CD45. Structure/function experiments in which wild-type or mutant CD45RA and CD45RO isoforms were expressed in CD45-deficient Jurkat cells revealed that the 19-aa insert is important for optimal CD45 function. The ability of both CD45RA and CD45RO to reconstitute CD3-mediated signaling based on measurement of calcium mobilization and mitogen-activated protein kinase activation was significantly decreased by deletion of the 19-aa insert. Mutation of four serine residues within the 19-aa insert to alanine affected CD45 function to a similar extent compared with that of the deletion mutants. These findings support the hypothesis that a physical interaction between the CD45 cytoplasmic domain and CK2 is important for post-translational modification of CD45, which, in turn, regulates its catalytic function.     

Statistical evaluation of inter- and intra-laboratory variations of the Ames test, as related to the genetic stability of Salmonella tester strains

A statistical analysis was performed on the data resulting from an international collaborative study of the Ames test according to a standardized experimental protocol, which involved the comparative testing of 4NQO (4 doses), in 3 separate experiments for each of the 38 participating laboratories, by using a common reference (R) culture and in-house laboratory (L) cultures of 5 strains of S. typhimurium. Despite some toxicity phenomena recorded at the highest dose of 4NQO, the majority of the dose-response curves in individual laboratories were linear on a bi-log scale and their mean values fitted a linear regression framework. Scattering of data around mean values of laboratories was Gaussian-like even at the highest dose of 4NQO, toxic effects being expressed as a dose-related increase of variance. A weighted least-square analysis could therefore take into account toxic effects without resorting to a sophisticated non-linear model incompatible with log transformation. Various analytical approaches--e.g. the weighted estimates of linear regression parameters, a multifactor (laboratory, experiment, dose, culture of each strain) analysis of variance with all the possible interactions, the assessment of correlations in individual laboratories and of coefficients of variation for induced and spontaneous mutability--could detect some statistically significant differences between L and R cultures. However, at a critical evaluation on an individual basis, only few of these differences, without any peculiar involvement of given strains, were convincing in view of the existence of real phenomena of genetic drift. Therefore, on the whole, the genetic drift of Salmonella tester strains appears to lend a negligible contribution to the considerable inter- and intra-laboratory variability detected in this study. With a background variability between replications averaging 26%, a dose-related variability was evident both between experiments (28-54%) and between laboratories (44-127%).     

Comparative evaluation of PCR assays for the robust molecular detection of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) can cause a very serious, often-fatal disease, namely paratuberculosis, in several animal species, especially ruminants. Recently, it has also been implicated in the pathogenesis of Infectious Bowel Disease of man. The aim of this study was to develop a molecular method for the routine detection and identification of MAP, from tissue samples of animal origin. The proposed assay would have to combine optimum performance and cost, with high reproducibility. To this goal, three laboratories in Greece and the Czech Republic undertook different parts of a study that involved evaluation of DNA extraction procedures, and PCR assays, for MAP detection. For DNA extraction we used one in-house, and one commercial method, and for the PCR we assessed a number of different assays, starting with the evaluation of primer specificity with an extended GenBank database search. Based on these results, we chose to assess a one-tube nested, 2 two-tube nested, and a single PCR assay, targeted to different genomic regions of the IS900 element. These four methods were applied on positive and negative control samples, consisted of pure bacterial cultures and formalin-fixed paraffin-embedded (FFPE) tissue samples collected from cattle with paratuberculosis and chickens with M. avium subsp. avium infection. Based on the criteria of reliability and cost, the procedure that performed better was the one-tube nested PCR assay combined with the in-house DNA extraction method. The agreement of the results obtained by the three collaborating laboratories indicates the reliability of the proposed assay even under different laboratory conditions.     

An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity

The Bhas 42 cell transformation assay is a sensitive short-term system for predicting chemical carcinogenicity. Bhas 42 cells were established from BALB/c 3T3 cells by the transfection of v-Ha-ras gene and postulated to have acquired an initiated state in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay is capable of detecting both tumor-initiating and tumor-promoting activities of chemical carcinogens. The full assay protocol consists of two components, the initiation assay and the promotion assay, to detect the initiating activity and the promoting activity, respectively. An international study was carried out to validate this cell transformation assay in which six laboratories from three countries participated. Twelve coded chemicals were examined in total and each chemical was tested by three laboratories. In the initiation assay, concordant results were obtained by three laboratories for eight out of ten chemicals and in the promotion assay, concordant results were achieved for ten of twelve chemicals. The positive results were obtained in all three laboratories with the following chemicals: 2-acetylaminofluorene was positive in both initiation and promotion assays; dibenz[a,h]anthracene was positive in the initiation assay; sodium arsenite, lithocholic acid, cadmium chloride, mezerein and methapyrilene hydrochloride were positive in the promotion assay. o-Toluidin hydrochloride was positive in the both assays in two of the three laboratories. d-Mannitol, caffeine and l-ascorbic acid were negative in both assays in all the laboratories, and anthracene was negative in both assays in two of the three laboratories except one laboratory obtaining positive result in the promotion assay. Consequently, the Bhas 42 cell transformation assay correctly discriminated all six carcinogens and two tumor promoters from four non-carcinogens. Thus, the present study demonstrated that the Bhas 42 cell transformation assay is transferable and reproducible between laboratories and applicable to the prediction of chemical carcinogenicity. In addition, by comparison of the present results with intra-laboratory data previously published, within-laboratory reproducibility using the Bhas 42 cell transformation assay was also confirmed.     

Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma

The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the interlaboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay coefficient of variance, limit of detection, limit of quantification, and recovery was evaluated. Limits of detection were at or below 1 ng/ml for the assayed proteins in 30 μl of plasma. Assay reproducibility was acceptable for verification studies, with median intra- and interlaboratory coefficients of variance above the limit of quantification of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope-labeled protein as an internal standard instead of stable isotope-labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-MRM-MS can be made reproducible across independent laboratories and has the potential to be adopted widely for assaying proteins in matrices as complex as plasma.