Selected Articles by Category
General Biomarker Research
General Biomarker Research
Cardiovascular Disease
Cancer
Diagnosis of alpha-1-antitrypsin deficiency: An algorithm of quantification, genotyping, and phenotyping
Author: Snyder MR;Katzmann JA;Butz ML;Yang P;Dawson DB;Halling KC;Highsmith WE;Thibodeau SN;
BACKGROUND: Laboratory testing in suspected alpha-1-antitrypsin (A1AT) deficiency involves analysis of A1AT concentrations and identification of specific alleles by genotyping or phenotyping. The purpose of this study was to define and evaluate a strategy that provides reliable laboratory evaluation of A1AT deficiency. METHODS: Samples from 512 individuals referred for A1AT phenotype analysis were analyzed by quantification, phenotype, and genotype. A1AT concentrations were measured by nephelometry. Phenotype analysis was performed by isoelectric focusing electrophoresis. The genotype assay detected the S and Z deficiency alleles by a melting curve analysis. RESULTS: Of the 512 samples analyzed, 2% of the phenotype and genotype results were discordant. Among these 10 discordant results, 7 were attributed to phenotyping errors. On the basis of these data we formulated an algorithm, according to which we analyzed samples by genotyping and quantification assays, with a reflex to phenotyping when the genotype and quantification results were not concordant. Retrospective analyses demonstrated that 4% of samples submitted for genotype and quantitative analysis were reflexed to phenotyping. Of the reflexed samples, phenotyping confirmed the genotype result in 85% of cases. In the remaining 15%, phenotyping provided further information, including identifying rare deficiency alleles and suggesting the presence of a null allele, and allowed for a more definitive interpretation of the genotype result. CONCLUSIONS: The combination of genotyping and quantification, with a reflex to phenotyping, is the optimal strategy for the laboratory evaluation of A1AT deficiency
Antibody multispecificity in immunoassay interference
Author: Levinson SS;
Recent findings indicate that many endogenous antibodies exhibit multispecificity. These antibodies exhibit a potential for interference with immunoassays. Antibodies that interfere with immunoassays have been called heterophile or heterophilic antibodies. The purpose of this review is: (1) to identify the nature of heterophile antibodies; (2) to delineate the processes that produce them; (3) to examine the mechanisms by which these antibodies cause interference; and (4) to explore how this information can be used to reduce immunoassay interference. In addition to producing specific antibodies, the process of antibody production gives rise to rudimentary antibodies that are polyspecific; e.g., the antigen-combining site has an affinity for antigens of different chemical composition. This process also generates idiotypic antibodies containing cross-reactive idiotopes. These antibodies along with rheumatoid factors, which are themselves polyspecific and rich in cross-reactive idiotopes, are inherent parts of the process of antibody production, and exhibit multispecificity. Mechanisms by which these antibodies cause immunoassay interference are outlined. These properties of antibodies may have substantial consequence in directing future assays toward greater clinical predictive value
False-positive immunoassay results: a multicenter survey of erroneous immunoassay results from assays of 74 analytes in 10 donors from 66 laboratories in seven countries
Author: Marks V;
BACKGROUND: Analytical interference in immunoassays can produce serious errors, but it is generally considered rare with modern analytical systems. METHOD: Blood was collected from 10 donors with illnesses known to be associated with rheumatoid factor. Immunoassays for 74 analytes were performed in 66 clinical laboratories. Each sample was measured in duplicate, and again in duplicate after the addition of a proprietary heterophil blocking reagent, with the laboratory's routinely used reagents and equipment. Reagents were typically supplied by the manufacturers of the closed analytical systems. Both competitive and sandwich immunoassays were investigated. RESULTS: Overall approximately 8.7% of the 3445 results were considered to be 'false positive', many of them seriously so. Twenty-one percent of the erroneously high results (1.8% of all results) were potentially misleading and were corrected by blocking reagent, although 49% of such seriously high results (4.2% of all results) were not. A further 39% of the false-positive results (2.6% of all results) would not necessarily have appeared likely to produce adverse clinical consequences but were substantially lowered by the addition of the blocking reagent. CONCLUSIONS: Random errors can occur with all types of immunoassays tested and can be difficult to identify even when repeated in another laboratory. Clinicians need to be aware of these limitations
Heterophile antibody interference in a multiplexed fluorescent microsphere immunoassay for quantitation of cytokines in human serum
Author: Martins TB;Pasi BM;Litwin CM;Hill HR;
While modern immunoassays provide sensitive and specific means for the quantitation of cytokines in biological fluids, heterophile antibodies are still a well-recognized cause of interference in the measurement of cytokines in these assays. We have developed a multiplexed fluorescent microsphere immunoassay for the simultaneous quantification of 10 cytokines in only 75 microl of serum. During the development of this multiplexed assay, the amount of assay interference due to heterophile antibodies was also determined, and methods for detecting heterophile interference and minimizing its effect were incorporated into the assay. Heterophile antibodies resulted in significantly elevated cytokine values compared to those of normal blood bank samples. These falsely elevated values, and thus the components of the assay the heterophile antibodies were binding to, were identified through the use of internal controls. This information was then used to design assay-specific blockers and absorbents that were shown to significantly reduce falsely elevated cytokine values while not affecting the standard and control values. The fluorescent multiplexed microsphere-based immunoassay can be used to quantitate multiple cytokines from a single sample and should be a useful tool in furthering our understanding of the role of cytokines in disease processes
The Human Plasma Proteome
Author: N.Leigh Anderson and Norman G.Anderson;
The human plasma proteome holds the promise of a revolutionin disease diagnosis and therapeutic monitoringprovided that major challenges in proteomics and relateddisciplines can be addressed. Plasma is not only the primaryclinical specimen but also represents the largestand deepest version of the human proteome present inany sample: in addition to the classical 'plasma proteins,'it contains all tissue proteins (as leakage markers) plusvery numerous distinct immunoglobulin sequences, and ithas an extraordinary dynamic range in that more than 10orders of magnitude in concentration separate albuminand the rarest proteins now measured clinically. Althoughthe restricted dynamic range of conventional proteomictechnology (two-dimensional gels and mass spectrometry)has limited its contribution to the list of 289 proteins(tabulated here) that have been reported in plasma todate, very recent advances in multidimensional surveytechniques promise at least double this number in thenear future. Abundant scientific evidence, from proteomicsand other disciplines, suggests that among these areproteins whose abundances and structures change inways indicative of many, if not most, human diseases.Nevertheless, only a handful of proteins are currently usedin routine clinical diagnosis, and the rate of introduction ofnew protein tests approved by the United States Food andDrug Administration (FDA) has paradoxically declinedover the last decade to less than one new protein diagnosticmarker per year. We speculate on the reasonsbehind this large discrepancy between the expectationsarising from proteomics and the realities of clinical diagnosticsand suggest approaches by which protein-diseaseassociations may be more effectively translated intodiagnostic tools in the future.