Biomarkers standardization, standard reference

The univariate response data on all standard biomarker data were analysed, ineluding analysis of variance for unbalaneed design, using Genstat v7.1 statistical software (VSN, 2003). In addition, a-priori pairwise t-tests were performed with the mean reference value, using the pooled variance estimate from the ANOVA. The real value data were not transformed. The average values for the KMBA and WOP biomarkers were not based on different flounder eaptured at the sites, but on replicate measurements of pooled liver tissue. The nominal response data of the immunohistochemical biomarkers (elassification of effects) were analysed by means of a Monte... 

As developed in Chapter 6, workplace biomarker targets also provide context and a frame of reference for communicating general-population biomonitoring results. However, this raises a number of communication issues, given that workplace biomarker criteria are not directly relevant to the general public, for the reasons described previously and because the standards may be established with a different level of health protection than would be suitable for the general public. 

The approaches described previously can be used to relate biomonitoring results to a reference population or to workplace exposures, but they do not evaluate the risk associated with the amount of a chemical found in the body. To do that, one needs to develop a relationship between biomarker concentration and toxic response, a relationship that is not commonly derived in standard toxicologic practice. The following sections outline methods for deriving such a relationship. The approaches include the ideal case of existing risk assessments based on biomarker-response relationships established in epidemiologic research. Lead and mercury are used as examples of cases in which exposure was quantified according to hair or blood biomarkers and dose-response associations were developed on this basis. 

The ultimate goal of an assay or an analytical procedure is to measure accurately a quantity or a concentration of an analyte, or to measure a specific activity, as in some assays for biomarkers. However, many activity assays, such as cell-based and enzyme activity assays, may not be very sensitive, may lack precision, and/or do not include the use of definitive reference standards. Assays based on measurements of physicochemical (such as chromatographic methods) or biochemical (such as ligand-binding assays) attributes of the analyte assume that these quantifiable characteristics are reflective of the quantities, concentration, or biological activity of the analyte. For the purpose of bioanalytical method validation, we will follow the recently proposed classifications for assay data by Lee et al. [4,5]. These classifications, as summarized below, provide a clear distinction with respect to analytical validation practices and requirements. 

In-study validation entails the routine monitoring of the quality control samples to determine whether the analytical method is performing consistently over time and whether data from a particular plate or run are acceptable. In addition, especially for biomarker assays, evaluation of parallelism using incurred samples is carried out to confirm the validity and suitability of the reference standard. 

At a minimum, documentation of the characterization and stability of a standard, such as a certificate of analysis (Co A) and/or a certificate of stability (CoS), is typically available from the suppliers. The certificate should be obtained and recorded. The quantity of reference standard is typically limited in commercial kits designed for research use, and it is not uncommon that the reference material values may differ substantially between lots and manufacturers [16]. Novel biomarkers rarely have established gold standards against which their potency and abundance can be calibrated. A comparison of available sources can be useful, and when validating an assay for advanced applications it is desirable to plan ahead to obtain and reserve a sufficient supply of the same reference material. The example in Fig. 6.5 compares three reference standard curves, each prepared from a concentrated stock solution from a commercial supplier, an in-house reference standard, and a commercial kit, respectively. The instrument responses (optical density, OD) were highest with the standard from the commercial stock, the lowest with the kit, while the in-house reference standard response was intermediate. In this case, either the same commercial stock or the in-house reference standard can be used throughout the clinical study. 

For a majority of biomarker assays, standard calibrators are prepared in an analyte-free alternative matrix instead of the de facto sample matrix of patient samples. For such methods, it is crucial to demonstrate that the concentration response relationship in the sample matrix is similar to that of the alternate matrix. Spike-recovery experiments with the reference standard may be inadequate to evaluate the matrix effect, as the reference standard may not fully represent the endogenous analyte. Instead, parallelism experiments are performed through serial dilutions of a high-concentration sample with the calibrator matrix. Multiple individual matrix lots (>3 lots) should be tested to compare lot-to-lot consistency. In the instance that the limited amounts of sample are available, apooled matrix strategy can be used with caution as discussed by Lee et al. [15]. The samples can be serially diluted with the standard matrix (standard... 

Defining the context of use for novel biomarkers in man represents an important area of collaborative research interest. Understanding reference ranges for novel DILI biomarkers in preclinical species and their evaluation in diverse healthy human populations and liver disease cohorts is an important area of investigation and question to address. Further areas of research focus should also be targeted toward the generation of robust cross species bioanalytical assays that are standardized or point-of-care tests in parallel with a comprehensive understanding of cross species differences in biomaiker expression, mechanisms of release, and clearance, distribution, and kinetics. 

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