Specificity biomarker assays

There is no clinical disease state that is pathognomonic for lead exposure. The neurotoxic effects and hematopoietic effects of lead are well recognized. The primary biomarkers of effect for lead are EP, ALAD, basophilic stippling and premature erythrocyte hemolysis, and presence of intranuclear lead inclusion bodies in the kidneys. Of these, activity of ALAD is a sensitive indicator of lead exposure (Hemberg et al. 1970 Morris et al. 1988 Somashekaraiah et al. 1990 Tola et al. 1973), but the assay can not distinguish between moderate and severe exposure (Graziano 1994). Sensitive, reliable, well-established methods exist to monitor for these biomarkers however, they are not specific for lead exposure. Therefore, there is a need to develop more specific biomarkers of effect for lead. Recent data... 

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. 

Methods for Biomarkers of Exposure. No methods were located for measuring any biomarkers of exposure to BCME. Although covalent adducts of BCME with cellular proteins or DNA have not yet been reported, development of sensitive and specific immunological assays for such adducts would provide a valuable means of detecting and perhaps quantifying human exposure levels. 

A variety of biomarkers have been shown to be valuable individually for one or several toxicant or disease situations. Few of these biomarkers have been systematically evaluated for the plethora of situations that might provoke false positive responses. Acceleration of the current pace of biomarker evaluation and qualification demands (a) the availability of panels of biomarker-assays that can be comparatively evaluated on well-defined common sample sets, (b) fit-for-purpose performance evaluation in controlled animal studies with carefully benchmarked histological endpoints and samples from well-defined focused clinical trial cohorts, and (c) ready availability of banked blood and urine sample archives from clinical trial populations with carefully documented morbidities such as the Framingham Heart Study,45 or the Drug-Induced Liver Injury Network (DILIN) prospective study,46 to name a few. Availability of such panels of validated biomarker assays and well-documented preclinical and clinical samples, as well as increased cooperation between animal model researchers and clinical researchers will enable individual biomarkers to be qualified for sensitivity of specifically defined adverse events, qualified for appropriate specificity using samples of defined benign events, and collected into panels that yield complementary information about the health and safety of animals and patients. 

Butoxyethanol has been shown to increase human plasma osmolality in vitro (Browning and Curry 1992). The osmolal gap is frequently used in the evaluation of a patient who has accidentally or intentionally ingested a glycol ether or related compound (Gijsenbergh et al. 1989). The presence of an elevated osmolal gap indicates the presence of significant blood levels of the toxic compound. This is not an indicator that is specific to 2-butoxyethanol or 2-butoxyethanol acetate and should be used in conjunction with other biomarker assays. 

A significant amount of LB A work, particularly in diagnostics and the burgeoning field of biomarker assays, employs LBAs in the form of manufacturer s kits. Chapter 7 focuses on considerations for the correct application of these kit assays, including the proper level of validation needed to support specific applications. 

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]. 

Careful understauding of the effects of each preanalytical variable on the biomarker data is complex and necessitates a staged approach conceptually similar to the fit-for-purpose analytical validation of a biomarker assay (see Chapter 41). In the early exploratory phase of biomarker investigation, standardization of procedures with a defined protocol for sample collection and handling will permit comparative interpretation and analysis of the data within study and/or between studies. Minimally, variables that should be experimentally evaluated to optimize sample collection for a specific biomarker will include matrix type, preservation... 

This is a complicated question but one that must be thought about. The number of single biomarker assays that are failing in the market is high, and the FDA in the United States has not approved a single biomarker assay in the last year. The reason for this is that in most cases the single biomarker assay cannot show the level of sensitivity and specificity necessary to be an effective diagnostic. 

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