Validation samples , biomarker

As for immunoassays for pharmaceutical proteins, in-study validation of biomarker assays should include one set of calibrators to monitor the standard curve as well as a set of QC samples at three concentrations analyzed in duplicate for the decision to accept or reject a specific run. Recommended acceptance criterion is the 6-4-30 rule, but even more lenient acceptance criteria may be justified based on statistical rationale developed from experimental data [14]. 

Biological, individual, and variations between individuals (such as gender, age, and nutrition) affect peptidomes and require careful consideration in order to find valid biomarkers. A few, equally important factors for successful proteomic biomarker research are high sample quality, high sensitivity, and reproducibility that depend on proper selection of the high quality samples. 

Until now, the composition of positive IHC controls resemble that of the patient sample. Pathologic discard tissue sections that express the biomarker in question are almost universally used as positive controls. Clinical laboratories bear the responsibility for identifying and validating the controls, typically from previous cases. An alternative QC material, which is especially popular for HER2 testing, is to use cell lines expressing the biomarker in question (as described in Chapter 6). Positive controls comprised of tissue sections or cell lines resemble patient samples in that they are cellular in nature. 

Microarrays have become robust, reliable research tools that enable researchers to screen for a multitude of parameters using minimal amounts of sample material. The acceptance of protein microarrays is growing constantly they have already been demonstrated to be useful tools in disease-related biomarker discovery. In addition, protein microarrays have been introduced into clinical trials in order to investigate the potential adverse effects of drug candidates. Depending on the number of validated disease-specific biomarkers, as well as on their therapeutic relevance, such assays are performed either on a protein microarray or a bead-based platform. 

Methods for Determining Biomarkers of Exposure and Effect. Section 2.6.1 reported on biomarkers used to identify or quantify exposure to diazinon. Some methods for the detection of the parent compound in biological samples were described above. The parent chemical is quickly metabolized so the determination of metabolites can also serve as biomarkers of exposure. The most specific biomarkers will be those metabolites related to 2-isopropyl-6-methyl-4-hydroxypyrimidine. A method for this compound and 2-(r-hydroxy-l -methyl)-ethyl-6-methyl-4-hydroxypyrimidine in dog urine has been described by Lawrence and Iverson (1975) with reported sensitivities in the sub-ppm range. Other metabolites most commonly detected are 0,0-diethylphosphate and 0,0-diethylphosphorothioate, although these compounds are not specific for diazinon as they also arise from other diethylphosphates and phosphorothioates (Drevenkar et al. 1993 Kudzin et al. 1991 Mount 1984 Reid and Watts 1981 Vasilic et al. 1993). Another less specific marker of exposure is erythrocyte acetyl cholinesterase, an enzyme inhibited by insecticidal organophosphorus compounds (see Chapter 2). Methods for the diazinon-specific hydroxypyrimidines should be updated and validated for human samples. Rapid, simple, and specific methods should be sought to make assays readily available to the clinician. Studies that relate the exposure concentration of diazinon to the concentrations of these specific biomarkers in blood or urine would provide a basis for the interpretation of such biomarker data. 

If an analytic method lacks sufficient specificity, chemical interferences will result in an erroneously high reported concentration. If a measured chemical is introduced as an artifactual contaminant during sample collection or analysis, reported concentrations will also be overestimated. For example, credibly estimating human exposure to phthalates was hindered by the difficulties involved in avoiding specimen contamination with these ubiquitous chemicals the problem was resolved by focus on the much less prevalent metabolic product, the phthalate half-ester (Silva et al. 2004). Alternatively, a chemical measured as a marker of exogenous exposure may be identical with a chemical formed by an unrelated endogenous metabolic pathway. In each of those cases, a rigorous laboratory-method validation should detect the problem before data are reported. More subtly, the measured biomarker of exposure may be chemically identical with a dietary... 

A carefully, comprehensively designed study facilitates study conduct. Two key elements of study conduct are sample collection and laboratory analysis. The choice of matrix depends on both theoretical and practical considerations. Those include the tissue most likely to represent the biomarker route of exposure and the ease of obtaining samples. Careful attention to collection and shipping from field to laboratory is a prerequisite for valid laboratory analysis. The choice of analytic laboratory methods is... 

Methods for Determining Biomarkers of Exposure and Effect. Excellent sensitive and selective methods are available for the qualitative and quantitative measurement of the parent compound, chlorobenzene after it is separated from its sample matrix. Methods need to be validated for chlorobenzene. 

Advances in petroleum characterization at the molecular structure level by GC-MS methods renewed interest in OSC. Within the past few years, at least one-thousand new and novel OSC that previously were not known to be present in petroleum and bitumens have been reported. Tentative molecular structures inferred from GC-MS and other techniques have been confirmed in many cases by synthesis of authentic reference-compounds. The difficult and time-consuming synthetic work has been crucial in validating many of the novel structures. Another key finding has been that immature bitumens and crude oils (samples that have not received significant thermal stress) differ markedly from the previously known OSC in that they have carbon-skeletons resembling ubiquitous biomarker hydrocarbons (e.g., n-alkanes, isoprenoid alkanes, steranes, and hopanes). This similarity, of course, suggests that the hydrocarbons and OSC have common biogenic precursors. 

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