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Digoxin concentrations were measured in serum samples from 102 patients with renal impairment who were receiving digoxin therapy. Many patients had values that differed widely on several currently available immunoassays, with differences as great as 2.9 ng/mL. In contrast, patients with normal renal function who were receiving digoxin had few discrepant results, with the largest difference being 0.5 ng/mL. We also assayed serum samples from 54 patients with renal impairment not on digoxin therapy and found that more than 60% of these digoxin-free patients had false-positive digoxin values on most assays. Our data suggest that a substance with digoxin-like immunoactivity is present in many patients with renal insufficiency. This substance may seriously compromise the accuracy and interpretation of digoxin concentration measurements. [ABSTRACT FROM AUTHOR]

Radiographic imaging is the current standard for monitoring progression of tumor-burden and therapeutic resistance in patients with metastatic melanoma. Plasma circulating tumor DNA (ctDNA) has shown promise as a survelience tool, but longitudinal data on the dynamics between plasma ctDNA concentrations and radiographic imaging is lacking. We evaluated the relationship between longitudinal radiographic measures of tumor burden and ctDNA concentrations in plasma on 30 patients with metastatic melanoma on systemic treatment. In 9 patients with no radiographic evidence of disease over a total of 15 time points, ctDNA concentrations were undetectable. In 21 patients with radiographic tumor burden, ctDNA was detected in 81 % of 58 time points. Plasma ctDNA concentrations demonstrated a modest positive correlation with total tumor burden (TTB) measurements (R 2 = 0.49, p < 0.001), with the greatest degree of correlation observed under conditions of progressive disease (PD) (R 2 = 0.91, p = 0.032). Plasma ctDNA concentrations were significantly greater at times of RECIST v1.1 progression (PD; 22.1 % ± 5.7 %) when compared to samples collected during stable disease (SD; 4.99 % ± 3.0 %) (p = 0.012); this difference was independent of total tumor burden (p = 0.997). Changes in plasma ctDNA showed a strong correlation with changes in TTB (R 2 = 0.88, p<0.001). These data suggest that measurements of plasma ctDNA during therapy are a better surrogate for responding versus non-responding disease compared to absolute tumor burden., Competing Interests: Declaration of competing interest The authors of the manuscript Corresponding ctDNA and tumor burden dynamics in metastatic melanoma patients on systemic treatment by Michael E. Egger(2), Evan Alexander(1), Tracy Van Meter(3), Maiying Kong(4,6), Aye Maung (4,6) Roland Valdes Jr.(1,7), Melissa Barousse Hall(5,8), and Mark W. Linder(1) declare no competing interest., (Copyright © 2024. Published by Elsevier Inc.)

The urocortins are polypeptides belonging to the corticotropin-releasing hormone family, known to modulate stress responses in mammals. Stress, whether induced physically or psychologically, is an underlying cause or consequence of numerous clinical syndromes. Identifying biological markers associated with the homeostatic regulation of stress could provide a clinical laboratory approach for the management of stress-related disorders. The neuropeptide, urocortin 3 (UCN3), and the corticotropin-releasing hormone receptor 2 (CRHR2) constitute a regulatory axis known to mediate stress homeostasis. Dysregulation of this peptide/receptor axis is believed to play a role in several clinical conditions including post-traumatic stress, sleep apnea, cardiovascular disease, and other health problems related to stress. Understanding the physiology and measurement of the UCN3/CRHR2 axis is important for establishing a viable clinical laboratory diagnostic. In this article, we focus on evidence supporting the role of UCN3 and its receptor in stress-related clinical syndromes. We also provide insight into the measurements of UCN3 in blood and urine. These potential biomarkers provide new opportunities for clinical research and applications of laboratory medicine diagnostics in stress management.

Background: Circulating tumor DNA (ctDNA) may complement radiography for interim assessment of patients with cancer., Objective: Our objective was to explore the relationship between changes in plasma ctDNA versus radiographic imaging among patients with metastatic melanoma., Methods: Using the Idylla system, we measured B-Raf proto-oncogene (BRAF) V600 ctDNA in plasma from 15 patients with BRAF V600E/K-positive primary tumors undergoing standard-of-care monitoring, including cross-sectional computed tomography (CT) imaging. BRAF V600 mutant allele frequency (%MAF) was calculated from the Idylla Cq values and directly measured using droplet digital polymerase chain reaction (ddPCR)., Results: The Idylla ctDNA assay demonstrated 91% sensitivity, 96% specificity, 91% positive predictive value, and 96% negative predictive value for the presence of > 93 mm metastatic disease. Qualitative ctDNA results corresponded to changes in RECIST (Response Evaluation Criteria in Solid Tumors) 1.1 status determined by CT imaging in 11 of 15 subjects (73%). Calculated %MAF results correlated with ddPCR (R 2 = 0.94) and provided evidence of progressive disease 55 and 97 days in advance of CT imaging for two subjects with persistently positive qualitative results., Conclusions: Overall, interim ctDNA results provided evidence of partial response or progressive disease an average of 82 days before radiography. This pilot study supports the feasibility of using the Idylla plasma BRAF V600 ctDNA assay as a complement to CT scanning for routine monitoring of therapeutic response. Somatic mutation quantification based on Cq values shows promise for identifying disease progression and warrants further validation.

The use of multiple medications is growing at an alarming rate with some reports documenting an average of 12-22 prescriptions being used by individuals ≥50 years of age. The indirect consequences of polypharmacy include exacerbation of drug-drug interactions, adverse drug reactions, increased likelihood of prescribing cascades, chronic dependence, and hospitalizations - all of which have significant health and economic burden. While many practical solutions for reducing polypharmacy have been proposed, they have been met with limited efficacy. This highlights the need for a new systematic approach for fine-tuning dispensing of medications. Pharmacogenetic testing provides an empirical and scientifically rigorous approach for guiding appropriate selection of medicines, with the potential to reduce unnecessary polypharmacy while improving clinical outcomes. The goal of this review article is to provide healthcare providers with an understanding of polypharmacy, its adverse effects on the healthcare system and highlight how pharmacogenetic information can be used to avoid polypharmacy in patients.

Liquid biopsy is a noninvasive dynamic approach for monitoring disease over time. It offers advantages including limited risks of blood sampling, opportunity for more frequent sampling, lower costs and theoretically non-biased sampling compared with tissue biopsy. There is a high degree of concordance between circulating tumor DNA mutations versus primary tumor mutations. Remote sampling of circulating tumor DNA can serve as viable option in clinical diagnostics. Here, we discuss the progress toward broad adoption of liquid biopsy as a diagnostic tool and discuss knowledge gaps that remain to be addressed.

Objectives: Immunoassays involving sample incubation followed by a wash step prior to introduction of labeled analyte are potentially subject to both positive and negative interference (bidirectional interference) by a competing ligand. We examine this phenomenon from a theoretical standpoint using a mathematical model for sequential-step immunoassays in the presence of interferent., Design & Methods: Competitive binding to antibody between analyte and interferent was modeled for sequential-step immunoassays. A primary assumption was that the ratio of affinity constants between the intended analyte and the interferent reflected the ratio of dissociation rate constants, with the higher dissociation rate constant for the lesser affinity ligand., Results: Relationships of parameters (relative affinity constants, relative concentrations) for analyte and interferent were determined for conditions in which bidirectional interference can occur, for both steady-state and non-steady-state sample incubation conditions. Non-steady state sample incubation conditions can enhance the effects of an interferent. Homogeneous assay formats utilizing labeled ligand without a wash step can also demonstrate bidirectional interference, but positive interference is favored under such formats., Conclusions: Model calculations demonstrate the theoretical basis for bidirectional interference in two-stage immunoassays. Results delineate constraints on conditions in which bidirectional interference can occur., (Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

There is a profound need in oncology to detect cancer earlier, guide individualized therapies, and better monitor progress during treatment. Currently, some of this information can be achieved through solid tissue biopsy and imaging. However, these techniques are limited because of the invasiveness of the procedure and the size of the tumor. A liquid biopsy can overcome these barriers as its non-invasive nature allows samples to be collected over time. Liquid biopsies may also allow earlier detection than traditional imaging. Liquid biopsies include the analysis of circulating tumor cells (CTCs), cell-free nucleic acid (cfNA), or extracellular vesicles obtained from a variety of biofluids, such as peripheral blood. In this review, we discuss different liquid biopsy types and how they fit into the current regulatory landscape.

This article introduces fundamental principles of pharmacogenetics as applied to personalized and precision medicine. Pharmacogenetics establishes relationships between pharmacology and genetics by connecting phenotypes and genotypes in predicting the response of therapeutics in individual patients. We describe differences between precision and personalized medicine and relate principles of pharmacokinetics and pharmacodynamics to applications in laboratory medicine. We also review basic principles of pharmacogenetics, including its evolution, how it enables the practice of personalized therapeutics, and the role of the clinical laboratory. These fundamentals are a segue for understanding specific clinical applications of pharmacogenetics described in subsequent articles in this issue., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Epigenetic testing, primarily in the form of DNA methylation analysis, is currently being used in healthcare settings to help identify and manage disease conditions and to develop and select drugs that specifically target epigenetic machinery. Yet, the clinical application of epigenetic analysis is still in its infancy. With a number of large-scale national and international epigenomic consortia projects in progress to identify tissue-specific epigenomes in normal and disease conditions, we are now poised for a new era of understanding disease processes based upon genetic changes that do not involve alterations to the DNA sequence. The developing epigenetic knowledge base will significantly advance the practice of personalized medicine and precision therapeutics. In this article, we provide a primer on the fundamentals of epigenetics with an emphasis on DNA methylation and review the prospective uses of epigenetic testing in advancing healthcare.

The measurement and characterization of circulating tumor cells (CTCs) hold promise for advancing personalized therapeutics. CTCs are the precursor to metastatic cancer and thus have the potential to radically alter patient treatment and outcome. Currently, clinical information provided by the enumeration of CTCs is limited to predicting clinical outcome. Other areas of interest in advancing the practice of pathology include: using CTCs for early detection of potential metastasis, determining and monitoring the efficacy of individualized treatment regimens, and predicting site-specific metastasis. Important hurdles to overcome in obtaining this type of clinical information involve present limitations in defining, detecting, and isolating CTCs. Currently, CTCs are detected using epithelial markers. The definition of what distinguishes a CTC should be expanded to include CTCs with heterogeneous phenotypes, and markers should be identified to enable a more comprehensive capture. Additionally, most methods available for detecting CTCs do not capture functionally viable CTCs. Retaining functional viability would provide a significant advantage in characterizing CTC-subtypes that may predict the site of metastatic invasion and thus assist in selecting effective treatment regimens. In this review we describe areas of clinical interest followed by a summary of current circulating cell-separation technologies and present limitations. Lastly, we provide insight into what is required to overcome these limitations as they relate to applications in advancing the practice of pathology and laboratory medicine.

We performed a randomised pilot trial of PerMIT, a novel decision support tool for genotype-based warfarin initiation and maintenance dosing, to assess its efficacy for improving warfarin management. We prospectively studied 26 subjects to compare PerMIT-guided management with routine anticoagulation service management. CYP2C9 and VKORC1 genotype results for 13 subjects randomly assigned to the PerMIT arm were recorded within 24 hours of enrolment. To aid in INR interpretation, PerMIT calculates estimated loading and maintenance doses based on a patient's genetic and clinical characteristics and displays calculated S-warfarin plasma concentrations based on planned or administered dosages. In comparison to control subjects, patients in the PerMIT study arm demonstrated a 3.6-day decrease in the time to reach a stabilised INR within the target therapeutic range (4.7 vs. 8.3 days, p = 0.015); a 12.8% increase in time spent within the therapeutic interval over the first 25 days of therapy (64.3% vs. 55.3%, p = 0.180); and a 32.9% decrease in the frequency of warfarin dose adjustments per INR measurement (38.3% vs. 57.1%, p = 0.007). Serial measurements of plasma S-warfarin concentrations were also obtained to prospectively evaluate the accuracy of the pharmacokinetic model during induction therapy. The PerMIT S-warfarin plasma concentration model estimated 62.8% of concentrations within 0.15 mg/l. These pilot data suggest that the PerMIT method and its incorporation of genotype/phenotype information may help practitioners increase the safety, efficacy, and efficiency of warfarin therapeutic management.

Background: Diagnostic informatics (DI) in the context of personalized medicine involves the integration of molecular information to provide "actionable" diagnostic and therapeutic strategies. In many cases, retrospective predictions of clinical outcomes affected by multiple genes are complicated by not having the relevant genes measured within the same study. Multiplicative effect modeling is a statistical method for estimating the net effect of ≥ 2 independent variables. The authors demonstrate a DI approach that uses multiplicative-effect modeling to combine genetic information from ≥ 2 independent studies to predict a net clinical outcome., Methods: As a hypothetical working model, 2 independent studies were selected each reporting on a unique genetic factor proposed to influence the risk of stent thrombosis (ST) among subjects treated with clopidogrel. A multiplicative effect model was used for developing a hypothesis regarding their combined influence on clinical outcome., Results: Application of multiplicative risk modeling yielded a revised estimated risk of outcomes based on combined genotype. In this scenario, combined genotype revised the categorical risk level (high versus low) estimated from single gene effects for 41.5% of the subjects. Further, the maximum relative risk based on single gene effects was increased from 4.54 to 7.84 based on combined genotype. The revised relative risk values in conjunction with combined genotype frequency estimates provides the data necessary to frame a trial hypothesis and conduct appropriate power analysis to estimate the number of subjects needed to test that hypothesis., Conclusions: This DI approach can be used to generate quantitative hypotheses on multiple gene effects derived from independent genotype studies. This approach is useful for estimating parameters needed in designing future studies to evaluate the net effect of ≥ 2 genetic variants on a common clinical endpoint.

Ill phases of bipolar illness are associated with abnormalities in ion regulation and intracellular ion concentrations. Previously, it has been reported that mania is characterised by lower circulating levels of ion regulating endogenous cardenolides, and that bipolar subjects lack the normal seasonal variation of these factors. Since endogenous cardenolides are elaborated in settings of extensive physical activity, euthymic bipolar and psychiatrically normal control subjects were asked to exercise to exhaustion. Plasma concentrations of endogenous cardenolides were measured at baseline, 60 min, peak exercise and post-recovery. Ouabain-like immunoreactive factor (OLF) was lower at baseline (0.005+/-S.D. 0.01 ng/mL in bipolar vs. 0.072+/-0.06 ng/mL in normal control subjects, P=0.019), lower at 60 min (0.007+/-S.D. 0.02 ng/mL in bipolar vs. 0.075+/-0.06 ng/mL in normal control subjects, P=0.029), and tended to be lower at peak exercise (0.009+/-S.D. 0.02 ng/mL in bipolar vs. 0.131+/-0.21 ng/mL in normal control subjects, P=0.15) in bipolar subjects compared to non-psychiatric controls. Other endogenous cardenolides did not vary significantly. The endogenous cardenolide, OLF, may be aberrantly controlled in bipolar illness.

Approximately 2-3% of all children in the United States suffer from obstructive sleep apnea (OSA). This condition is characterized by repeated events of partial or complete obstruction of the upper airways during sleep leading to recurring episodes of hypercapnia, hypoxemia, and arousal throughout the night as well as snoring, which afflicts 7-10% of all children. Since clinical history and physical examination are unreliable in the differentiation between children with OSA and children with primary snoring (PS) who have no apparent alteration in sleep architecture, current diagnostic approaches for OSA require an overnight sleep study (ONP). ONP is onerous, relatively unavailable, labor intensive, and inconvenient, leading to long waiting periods and unnecessary delays in diagnosis and treatment. Development of noninvasive biomarker(s) capable of reliably distinguishing children with PS from those with OSA would greatly facilitate timely screening and diagnosis of OSA in children. Therefore, we hypothesized that proteomic strategies in the urine may permit the identification of biomarker(s) that reliably screen for OSA. In this study, time-of-flight mass spectrometry was used to profile proteins in the first morning void urines from children. We discovered that urocortins are increased in OSA and provide a noninvasive approach for quick and convenient diagnosis otf OSA in snoring children.

Background: The application of pharmacogenetic results requires demonstrable correlations between a test result and an indicated specific course of action. We developed a computational decision-support tool that combines patient-specific genotype and phenotype information to provide strategic dosage guidance. This tool, through estimating quantitative and temporal parameters associated with the metabolism- and concentration-dependent response to warfarin, provides the necessary patient-specific context for interpreting international normalized ratio (INR) measurements., Methods: We analyzed clinical information, plasma S-warfarin concentration, and CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) and VKORC1 (vitamin K epoxide reductase complex, subunit 1) genotypes for 137 patients with stable INRs. Plasma S-warfarin concentrations were evaluated by VKORC1 genotype (-1639G>A). The steady-state plasma S-warfarin concentration was calculated with CYP2C9 genotype-based clearance rates and compared with actual measurements., Results: The plasma S-warfarin concentration required to yield the target INR response is significantly (P < 0.05) associated with VKORC1 -1639G>A genotype (GG, 0.68 mg/L; AG, 0.48 mg/L; AA, 0.27 mg/L). Modeling of the plasma S-warfarin concentration according to CYP2C9 genotype predicted 58% of the variation in measured S-warfarin concentration: Measured [S-warfarin] = 0.67(Estimated [S-warfarin]) + 0.16 mg/L., Conclusions: The target interval of plasma S-warfarin concentration required to yield a therapeutic INR can be predicted from the VKORC1 genotype (pharmacodynamics), and the progressive changes in S-warfarin concentration after repeated daily dosing can be predicted from the CYP2C9 genotype (pharmacokinetics). Combining the application of multivariate equations for estimating the maintenance dose with genotype-guided pharmacokinetics/pharmacodynamics modeling provides a powerful tool for maximizing the value of CYP2C9 and VKORC1 test results for ongoing application to patient care.

This article demonstrates how a dynamic clinical-support tool can guide individualized drug therapy. We use the drug warfarin as a model to demonstrate how pharmacogenetics when combined with fundamental principles of pharmacokinetic and pharmacodynamics can provide a powerful decision-support tool to optimize personalized therapeutics.

This article provides an introduction to the fundamental principles of pharmacokinetics (PK) and pharmacodynamics (PD) as they apply to understanding the application of pharmacogenetics (PGx) in a clinical setting. PGx establishes connections between the disciplines of pharmacology and genetics. As such, one functional component of PGx involves establishing relationships between phenotypes and genotypes with respect to predicting the response of medications in individual patients. The article begins by describing each of the concepts, followed by discussing the clinical utility of PGx and pharmacodynamics in a laboratory medicine setting; it then makes a link with the evolving field of PGx from the perspective of clinical laboratory medicine. Laboratory medicine serves as a catalyst for transitioning PGx into clinical settings, and as such, the article concludes by describing the future role of clinical laboratories in the application of PGx to patient care.

Digoxin-like immunoreactive factor (DLIF) and ouabain-like factor (OLF) are the mammalian counterparts to the plant-derived cardiotonic steroids digoxin and ouabain. Compelling evidence indicates that the cardiotonic steroids may have anticancer properties. Recent evidence indicates that low (nanomolar) concentrations of DLIF selectively induce cell death in transformed cells, while sparing normal cells, and is even more potent than the plant-derived compounds. The discovery that these endogenous molecules may play a role in the regulation of cancer cell proliferation provides a potentially new paradigm for the physiologic role of DLIF and OLF. In addition, the possible use of digoxin itself as a therapeutic agent in cancer has been explored, and evidence suggests that its conversion to dihydrodigoxin may be involved in regulating anticancer activity. The mechanism(s) for the pro-apoptotic property of these compounds is not known. In this brief review, we will discuss the proposed mechanism of action of digoxin, ouabain, DLIF, and OLF as anticancer compounds and discuss the effects that metabolic conversion to their dihydro-derivatives may have on this activity. From the perspective of therapeutic drug monitoring, these findings suggest some potential new challenges in the need to measure concentrations of digoxin and dihydrodigoxin as well as their endogenous counterparts DLIF and OLF in serum.

Objectives: Polymorphism in the genes for cytochrome (CYP)2C9 and the vitamin K epoxide reductase complex subunit 1 (VKORC1) affect the pharmacokinetics and pharmacodynamics of warfarin. We developed and validated a warfarin-dosing algorithm for a multi-ethnic population that predicts the best dose for stable anticoagulation, and compared its performance against other regression equations., Methods: We determined the allele and haplotype frequencies of genes for CYP2C9 and VKORC1 on 167 Caucasian, African-American, Asian and Hispanic patients on warfarin. On a subset where complete data were available (n=92), we developed a dosing equation that predicts the actual dose needed to maintain target anticoagulation using demographic variables and genotypes. This regression was validated against an independent group of subjects. We also applied our data to five other published warfarin-dosing equations., Results: The allele frequency for CYP2C9*2 and *3 and the A allele for VKORC1 3673 was similar to previously published reports. For Caucasians and Asians, VKORC1 SNPs were in Hardy-Weinberg linkage equilibrium. Some VKORC1 SNPs among the African-American population and one SNP among Hispanics were not in equilibrium. The linear regression of predicted versus actual warfarin dose produced r-values of 0.71 for the training set and 0.67 for the validation set. The regression coefficient improved (to r=0.78 and 0.75, respectively) when rare genotypes were eliminated or when the 7566 VKORC1 genotype was added to the model. All of the regression models tested produced a similar degree of correlation. The exclusion of rare genotypes that are more associated with certain ethnicities improved the model., Conclusion: Minor improvements in algorithms can be observed with the inclusion of ethnicity and more CYP2C9 and VKORC1 SNPs as variables. Major improvements will likely require the identification of new gene associations with warfarin dosing.

Cytochrome P450 2C9 (CYP4502C9) genotyping is useful in dosage adjustments for several critical drug therapies, including warfarin. Potential interference compromising these genotyping results could lead to inappropriate dose adjustments that may result in adverse drug reactions. During routine clinical CYP4502C9 genotyping using multiplex allele-specific primer extension, an ambiguous result was obtained for determination of the CYP2C9 430C>T substitution, which defines the CYP2C9*2 allele. In this one patient sample submitted for CYP2C9 genotyping, the ratio for the variant 430T allele signal to the total signal (C+T alleles) was 0.29. This is above the expected ratio to be classified as wild-type (<0.15) and below the minimum expected ratio (>0.3) when the genotype is heterozygous at the 430 position. The mean fluorescence intensity for the 430C allele was consistent with that observed in subjects who are heterozygous at this nucleotide position. However, the corresponding signal for the 430T allele indicated the absence of the CYP2C9*2 allele. This suggests the assay was not able to determine the correct nucleotide at position 430 for one of the two alleles in this patient. Subsequent sequencing to investigate the allele-specific primer extension failure revealed the presence of a rare C>T nucleotide substitution at position 429. We tested this subject's CYP2C9 genotype using AvaII restriction endonuclease digestion and found that this rare substitution causes false-positive identification of the CYP2C9*2 allele when using this method. We developed a DpnII endonuclease digestion assay to specifically detect the CYP2C9 429C>T substitution and tested 100 randomly selected samples obtained from unrelated individuals. The 429C>T polymorphism was not identified in this sample set, which indicates an allele frequency of less than 2.0% (95% confidence interval, 0.0-1.8%) in the general population. Despite the rarity of this polymorphism, it has important implications for the accuracy of assays using allele-specific primers and the Ava II restriction endonuclease, when it occurs, which are two common methods currently applied for detecting the presence of the CYP2C9*2 allele.

Background: CYP2C9 polymorphisms are associated with decreased S-warfarin clearance and lower maintenance dosage. Decreased expression of VKORC1 resulting from the -1639G>A substitution has also been implicated in lower warfarin dose requirements. We investigated the additional contribution of this polymorphism to the variance in warfarin dose., Methods: Sixty-five patients with stable anticoagulation were genotyped for CYP2C9 and VKORC1 with Tag-It allele-specific primer extension technology. Plasma S-warfarin concentrations and warfarin maintenance dose were compared among patients on the basis of the VKORC1 -1639G>A genotype., Results: Eighty percent of CYP2C9*1/*1 patients stabilized on <4.0 mg/day warfarin had at least 1 VKORC1 -1639A allele. Mean warfarin doses (SD) were 6.7 (3.3), 4.3 (2.2), and 2.7 (1.2) mg/day for patients with the VKORC1 -1639GG, GA, and AA genotypes, respectively. Steady-state plasma concentrations of S-warfarin were lowest in patients with the VKORC1 -1639AA genotype and demonstrated a positive association with the VKORC1 -1639G allele copy number (trend P = 0.012). A model including VKORC1 and CYP2C9 genotypes, age, sex, and body weight accounted for 61% of the variance in warfarin daily maintenance dose., Conclusions: The VKORC1 -1639A allele accounts for low dosage requirements of most patients without a CYP2C9 variant. Higher plasma S-warfarin concentrations corresponding to increased warfarin maintenance dosages support a hypothesis for increased expression of the VKORC1 -1639G allele. VKORC1 and CYP2C9 genotypes, age, sex, and body weight account for the majority of variance in warfarin dose among our study population.

Background: Plant-derived cardenolides reportedly possess anticancer properties in human leukemic cells via selective induction of apoptosis, cell cycle arrest, and differentiation. Selective induction of apoptosis with mammalian-derived digoxin-like immunoreactive factor (DLIF) could provide new strategies for anticancer drug development or the identification of biomarkers for cancer. We investigated whether DLIFs selectively induce apoptosis in human lymphoblastic leukemic cells., Methods: We compared the relative potencies of digoxin, ouabain, and DLIF on induction of programmed cell death in Jurkat cells (an acute T-leukemic cell line), K-562 (a myelogenous leukemia cell line), and nonpathologic human peripheral blood mononuclear cells (PBMCs). Apoptosis was measured by flow cytometry with the annexin V/propidium iodide method., Results: Digoxin and ouabain induced apoptosis in Jurkat cells [digoxin 50% inhibitory concentration (IC(50)), 24 nmol/L; ouabain IC(50), 26 nmol/L]. Neither digoxin nor ouabain induced apoptosis in K-562 cells or PBMCs. DLIF was more potent (IC(50), 1.9 nmol/L) and >2-fold more effective than digoxin or ouabain at inducing maximum apoptosis in Jurkat cells. The IC(50) values in the apoptosis assays were >100-fold lower (DLIF) and 20-fold lower (digoxin and ouabain) than the IC(50) required for Na(+)- and K(+)-dependent ATPase (DLIF, 200 nmol/L; digoxin, 910 nmol/L; ouabain, 600 nmol/L)., Conclusion: DLIF selectively induces apoptosis in a human acute T-cell lymphoblastic leukemia cell line but not in K-562 cells or PBMCs. These data suggest a new physiological role for these endogenous hormone-like factors.

Warfarin is the most commonly prescribed oral anticoagulant for the treatment and prevention of thromboembolic events. The correct maintenance dose of warfarin for a given patient is difficult to predict, the drug carries a high risk of toxicity, and variability among patients means that the safe dose range differs widely between individuals. Recent pharmacogenetic studies indicate that the routine incorporation of genetic testing into warfarin therapy protocols could substantially ease both the financial and health risks currently associated with this treatment. In particular, the variability in warfarin dose requirement is now recognized to be due, in large part, to polymorphisms in two genes: cytochrome P450 2C9 and the vitamin K epoxide reductase complex subunit 1. The development of algorithms that integrate all of the relevant genetic and physical factors into comprehensive, individualized predictive models for warfarin dose could be used to translate the results of pharmacogenetic testing into actionable clinical application.

Urinary trypsin inhibitors (uTi) suppress serine proteases during inflammation. After liberation from proinhibitors (P-alpha-I and I-alpha-I) by the white blood cell (WBC) response, uTi readily pass through the kidneys into urine. A key uTi, bikunin, is attached to O-linked and N-linked glycoconjugates. Recently, uTi inhibitors, called uristatins, were found to lack the O-linked glycoconjugates. Monoclonal antibodies were produced using purified uristatin and screened for binding differences to uristatin, bikunin, P-alpha-I, and I-alpha-I. Antibody-binding patterns were characterized using immunoaffinity binding onto protein-chip surfaces and analysis by Surface Enhanced Laser Desorption/Ionization mass spectrometry (SELDI), using specimens from patients and from purified uTi standards. Antibodies were developed and used in an enzyme-linked immunosorbent assay (ELISA) method for uTi measurement in urine and plasma specimens. ELISA was performed on specimens from normal, presumed healthy, controls and from patients who had been screened for inflammation using a high sensitivity C-reactive protein (CRP) test and a complete blood count (CBC). Polyclonal antibody against uTi showed cross-reactivity with the Tamm-Horsfall protein (THP) and with proinhibitors. Screening of anti-uTi monoclonal antibodies (Mab) revealed antibodies that did not cross-react with either of the above, thus providing a tool to measure both uristatin and bikunin in urine with Mab 3G5 and in plasma with Mab 5D11. The monoclonal antibody 5D11 cross-reacts with specific N-linked glycoconjugates of uristatin present in plasma. In ca 96% of healthy adults, uTi were present at <12 mg/l in urine and <4 mg/l in plasma. We also found that patients with an inflammation and a CRP of >2.0 mg/l had higher urinary concentrations of uTi than the control population in every subject. Free uristatin and bikunin pass readily into urine and are primarily bound to heavy chains that constitute the proinhibitor form in plasma.

Inflammatory processes, such as phagocytosis, coagulation, and vascular dilation, promote the release of serine proteases by neutrophils, macrophages, mast cells, lymphocytes, and the epithelial or endothelial cells. These proteases further facilitate the release of inflammatory cytokines and growth factors as well as take part in signal-cell proliferation through protease-activated receptors (PARs). Controlling the action of this cascade is necessary to prevent further damage to the normal tissues. One of the main anti-inflammatory response mediators is bikunin (Bik) that is responsible for inhibiting the activity of many serine proteases such as trypsin, thrombin, chymotrypsin, kallikrein, plasmin, elastase, cathepsin, Factors IXa, Xa, XIa, and XlIa. During the acute-phase response, Bik is released into plasma from proinhibitors primarily due to increased elastase activity. Bik is a glycoprotein, also referred to as urinary trypsin inhibitor, which in plasma inhibits the trypsin family of serine proteases by binding to either of the two Kunitz-binding domains. Bik also accumulates in urine. In conditions such as infection, cancer, tissue injury during surgery, kidney disease, vascular disease, coagulation, and diabetes, the concentrations of Bik in plasma and urine are increased. Several trypsin inhibitory assays for urine and immunoassays for both blood and urine have been described for measuring Bik. In addition to presenting the synthesis, structure, and pathophysiology of Bik, we will summarize various diagnostic approaches for measuring Bik. Analysis of Bik may provide a rapid approach in assessing various conditions involving the inflammatory processes.

Background: Ligand binding is commonly analyzed using various immunoassays that are generally time-consuming and some may require secondary antibodies or gel electrophoresis which are also time-consuming and sometimes subjective. We introduced various examples for a more rapid approach using pre-activated surface chips which are analyzed by surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Specific applications presented in this study include immobilization of antigen, antibody or oligo DNA on pre-activated chips with subsequent identification of the binding antibodies, antigens or DNA binding proteins to demonstrate the universal utility of this novel approach., Methods: BSA-digoxin conjugate (BSA-Dig), anti-digoxin antibody, anti-urinary trypsin inhibitor (uTi) antibody, or a double stranded oligo nucleotide based on the nucleotide sequence between -91 and -10 of the human CYP 450 2E1 promoter were immobilized on the Ciphergen pre-activated surface chips. Anti-digoxin antibody, BSA-digoxin conjugate, uTi, and CYP450 2E1 promoter binding protein were captured on the chip and identified by SELDI-TOF MS., Results: A protein with 141kDa was identified from anti-digoxin serum using BSA-Dig chips. This binding was competitively inhibited by addition of digoxin. Using anti-digoxin antibody, a peak at approximately 66kDa was detected in the preparation of BSA-Dig. This peak was also inhibited by free digoxin, suggesting BSA-Dig is detected. uTi fragments with approximately 3kDa to approximately 30kDa in the standard and urine samples were captured on the chip by anti-uTi antibody. Finally, we identified a 95-kDa CYP 450 2E1 promoter binding protein in HeLa cells nuclear extracts., Conclusions: Bioaffinity SELDI-TOF MS is a powerful and versatile approach for analysis of ligands. It eliminates tracer-labeled secondary antibodies and allows for determination of molecular weights of binding proteins and their ligands directly. This approach may also be considered for the detection of enzymes, receptors, or any other specific ligands.

Background: Genotyping of N-acetyltransferase-2 (NAT2) is useful in predicting the risk for toxicity of NAT2 substrates. Current methods cannot detect the 7 most important single-nucleotide variations in NAT2 simultaneously in 1 tube., Methods: We developed an assay that uses allele-specific primer extension (ASPE) and microsphere hybridization for the simultaneous detection of 7 single-nucleotide variations in NAT2. Using 12 samples previously genotyped by a TaqMan-based assay for method development and as positive controls, we amplified the genetic locus of NAT2 comprising the single-nucleotide variations of interest by PCR and then performed ASPE with allele-specific primers and biotinylated dCTP followed by bead hybridization and streptavidin-R-phycoerythrin binding. Genotypes were determined according to the allele-specific fluorescent signal ratios., Results: The mean (SD) allelic ratios for homozygous common, heterozygous variant, and homozygous variant NAT2 genotypes were 0.0394 (0.0113) (n = 80), 0.4372 (0.0270) (n = 148), and 0.9331 (0.0127) (n = 325). The assay had 100% (95% confidence interval, 99%-100%) within-run reproducibility for 12 samples repeated 6 times and 100% (98%-100%) between-run reproducibility for a 5-sample subset run on 6 different days. NAT2 genotypes of 30 blinded samples determined by this assay were 100% (98%-100%) concordant with results obtained using the TaqMan method., Conclusions: The developed assay can simultaneously determine single-nucleotide variations in NAT2. The assay demonstrates no overlap in allele-specific signal ratios between homozygous common, heterozygous, and homozygous variant and shows agreement with a reference method and reproducibility of genotype identification.

Obstructive sleep apnea (OSA) is a major public health problem affecting approximately 2% to 3% of children. However, snoring, the cardinal symptom of OSA, affects at least 5-fold more children, such that evaluation by overnight polysomnography (ONP) is required for the diagnosis. ONP is laborious, expensive, and relatively unavailable to children. Proteomic mass spectrometry coupled with bioinformatic tools provide valuable means for discovery of new biomarkers in serum for a variety of human disorders. The possibility exists that snoring children with and without OSA may exhibit different protein expression profiles in serum that could be useful in the development of novel diagnostic tools for this condition. The proteomic patterns of 20 children with OSA and of 20 children with habitual primary snoring but no evidence of OSA (HS) were evaluated using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Linear discriminative analysis identified three differentially regulated proteins with molecular masses of 5896, 3306, 6068 Da that were capable of diagnosing OSA with 93% sensitivity and 90% specificity. Thus, the proteomic signatures of sera from children with OSA differ from those of HS who do not fulfill the current criteria for treatment. Identification and sequencing of those differentially expressed proteins discovered through proteomic strategies may lead to future development of serum-based diagnostic tests for OSA in snoring children.

Bioaffinity mass spectrometry is a novel technology for analysis of binding proteins and their ligands. In this review, we introduce the concepts and principles of bioaffinity surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Various preactivated chip types and several approaches for binding of ligands or their binders to the chips are discussed. We also provide specific examples for the use of this technology for screening antibodies, analyzing ligands, glycoconjugates, protein-protein inter-actions, and DNA (RNA) binding proteins. In pursuit of developing new tests or studies of mechanism of drug action in therapeutic drug monitoring practice, this technology may provide a more rapid approach for ligand-binder studies.

Background: Digoxin-like immunoreactive factors (DLIFs) are endogenous mammalian cardenolides with structural features similar to those of the plant-derived digitalis compounds. DLIFs and their structurally related forms (Dh-DLIFs) may serve as effectors of ion-transport activity mediated by their interaction with Na,K-ATPase and thus play a role as a new hormonal axis. Although some evidence implicates the adrenal gland as a tissue source for the DLIFs, little is known about the biosynthetic pathway producing these compounds. We now demonstrate de novo biosynthesis of DLIF by incorporation of radioactive carbon ((14)C) into the structures of both DLIF and Dh-DLIF., Methods: We used a combination of reversed-phase HPLC techniques to separate the radioactive DLIF components after incorporation of (14)C into their structure by use of either [1,2-(14)C]acetic acid or [4-(14)C]cholesterol as precursors and a Y-1 mouse adrenocortical tumor cell line. We also stimulated and suppressed production of steroidogenesis by use of cAMP analogs and Mevastatin, respectively, to demonstrate the dependence of DLIF production on the cholesterol-dependent biosynthetic pathway. A combination of chromatographic mobility, immunoassays specific for digoxin and dihydrodigoxin, and deglycosylation using 5-sulfosalicylic acid were used to identify the DLIF and Dh-DLIF components., Results: With cholesterol as precursor, the cells produced DLIF (7.5 mCi/mmol) with a labeling efficiency of 10%, whereas with acetate the cells produced DLIF (72.2 mCi/mmol) with a labeling efficiency of 0.08% of the total DLIF produced. The radiolabeled DLIF and Dh-DLIF molecules had identical chromatographic mobilities and stoichiometric removal of sugars as the previously characterized DLIFs isolated from different mammalian species and tissues. With radioactive cholesterol as precursor, the (14)C was incorporated into the DLIF-genin portion of the compounds and not the sugars. Interestingly, treatment of Y-1 cells with 8-bromoadenosine 3':5'-cAMP to stimulate steroidogenesis did not increase production of DLIF or Dh-DLIF but did increase production of progesterone. Mevastatin (5 micromol), an inhibitor of the enzyme hydroxymethylglutaryl-CoA reductase and thus of cholesterol biosynthesis, gave an 85% decrease in the production of (14)C-DLIF and progesterone, but only a modest 15% decrease in (14)C-Dh-DLIF production., Conclusions: These data demonstrate that the adrenal cell has the cellular machinery necessary for de novo biosynthesis of DLIF and Dh-DLIF starting from a simple carbon pool and also support the concept that cholesterol is a major precursor of the DLIF compounds. This cell culture model provides a source of radiolabeled DLIF compounds for future experimental work.

Background: We determined the diagnostic value of the trypsin inhibitor, uristatin, that is commonly found in urine and plasma in patients with infections or inflammations of any kind., Methods: We collected urine specimens from patients with infections of the urinary or upper respiratory tract and from healthy controls. We also collected blood from patients with a likely upper respiratory tract infection and healthy controls. A bacterial count of >10(5) organisms/ml in urine was considered to represent infection rather than contamination., Results: The uristatin dipstick test in urine showed acceptable negative predictive values (NPV of up to 93%) for patients without infection or inflammation. Here, the dipsticks could eliminate some urine cultures. For those with infection or inflammation, the positive predictive values (PPV) of the dipsticks were lower (up to 57%). Including the leukocyte esterase and nitrite values increased the PPV of the dipsticks for those with disease., Conclusions: The uristatin strip was more accurate than the leukocyte and nitrite dipsticks for predicting upper respiratory infections (URI) and C-reactive protein for those with infection or inflammation. The uristatin dipstick was able to detect both the bikunin and uristatin inhibitors.

Background: Heart failure (HF) is a devastating disease with increasing prevalence in elderly populations. One-half of all patients die within 5 years of diagnosis. The annual cost of treating patients with HF in the US is more than $20 billion, which is estimated to be greater than that of myocardial infarction and all cancers combined. Given the complex pathophysiology and varied manifestations of HF, interest has intensified in developing biological markers to predict susceptibility and aid in the early diagnosis and management of this disease., Methods: We searched Medline via Ovid for studies published during the period 1966-2003 regarding various biomarkers suggested for HF. Our review focused on developing strategies for discovering and using new biomarkers, particularly those potentially linked to pathophysiologic mechanisms. We also point out strategic advantages, limitations, and methods available for measuring each of the currently proposed markers., Results: Biomarkers reviewed include those released from the heart during normal homeostasis (natriuretic peptides), those produced elsewhere that act on the heart (endogenous cardiotonic steroids and other hormones), and those released in response to tissue damage (inflammatory cytokines). The concept of using a combination of multiple markers based on diagnosis, prognosis, and acute vs chronic disease is also discussed. In view of recent advances in our understanding of molecular biochemical derangements observed during cardiac failure, we consider the concept of myocardial remodeling and the heart as part of an endocrine system as strategies., Conclusion: Strategically, biomarkers linked to mechanisms involved in the etiology of HF, such as dysregulation of ion transport, seem best suited for serving as early biological markers to predict and diagnose disease, select therapy, or assess progression.

In this multicenter evaluation, the VERSANT HCV RNA 3.0 Assay (bDNA) (Bayer Diagnostics, Tarrytown, N.Y.) was shown to have excellent reproducibility, linearity, and analytical sensitivity across specimen collection matrices (serum, EDTA, ACD-A), and hepatitis C virus (HCV) genotypes 1 to 6. The VERSANT HCV bDNA Assay has a reportable range of 615 to 7690000 (7.69 x 10(6)) IU/ml. The total coefficient of variation (CV) ranged from 32.4% at 615 IU/ml to 17% at 6.8 x 10(6) IU/ml. The assay was linear across the reportable range. Analytical specificity of 98.8% was determined by testing 999 specimens from volunteer blood donors. Evaluation of HCV genotypes using RNA transcripts of representative clones of 1a, 1b, 2a, 2b, 2c, 3a, 4a, 5a, and 6a and patient specimens showed that the largest difference between genotype 1, upon which the assay is standardized, and non-1 genotypes was within 1.5-fold. Testing of potentially interfering endogenous substances and exogenous substances and conditions found no interference in HCV-positive or HCV-negative specimens except for unconjugated bilirubin at concentrations of >or=20 mg/dl and protein at concentrations of >or=9 g/dl. Biological variability was estimated from 29 clinically stable individuals not on HCV therapy who were tested weekly over an 8-week period. The combined estimate of total (biologic plus assay) variability was 0.15 log(10) standard deviation (CV, 36.1%), a fold change of 2.6. Thus, the observed fold change between any two consecutive HCV RNA measures is expected to be less than 2.6-fold (equivalent to 0.41 log(10) IU/ml) 95% of the time in clinically stable individuals.

Urinary trypsin inhibitor (uTi) is a product of elastase-mediated degradation of interleukin-alpha-inhibitor (I-alpha-I). Its activity increases in the urine of patients with a malignancy, inflammation, or infection, or in late pregnancy. The objective of this study was to compare the sensitivity of uTi in urine with that of serum quantitative C-reactive protein (CRP) for diagnosing infection, as indicated by white cell response and clinical assessment. Ninety controls and 171 patients with various systemic infections were enrolled. We measured uTi enzymatically on a Cobas Fara (Roche Diagnostics). Patients were separated into bacterial, probable bacterial, viral, or probable viral groups based on the results of a complete blood count with differential (CBC), urinalysis (UA), and clinical assessment. In the bacterial (n=70) and control (n=90) groups, the uTi values (mean+/-SE) were 25.3+/-3.1 mg/L and 2.8+/-0.8 mg/L, respectively. uTi (at 2.7 mg/L) had a diagnostic sensitivity of 91% and specificity of 82% (AUC=0.889), whereas CRP (at a cutoff of 10 mg/L) had a sensitivity and specificity of 82% and 96%, respectively (AUC=0.921). As a marker of infection (positive in both bacterial and viral groups), uTi had a sensitivity of 91% (AUC=0.884) vs. 89% (AUC=0.828) for CRP. Our data indicate that uTi has sufficient clinical sensitivity for screening systemic infections, and may have diagnostic value as a noninvasive test., ((c) 2004 Wiley-Liss, Inc.)

The CYP2E1*7B allele is defined by two nucleotide sequence polymorphisms, -71G>T and -333T>A. The CYP2E1 promoter sequence flanking the -71G nucleotide is consistent with a gamma-interferon activated sequence. Inflammation and interferon (IFN)-gamma suppress expression of CYP2E1 in vivo; however, the exact mechanism is not known. The objectives of this study were to determine whether the CYP2E1 promoter is regulated by IFN-gamma and to examine the influence of the nucleotide substitutions on this function. Treatment of HepG2 cells with IFN-gamma, after transient transfection with a luciferase reporter gene bearing the native CYP2E1 (-71G) promoter sequence resulted, in a dose-dependent reduction of luciferase activity. In contrast, no suppression was observed in cells transfected with the *7B allele promoter (-333A and -71T) nor a CYP2E1 plasmid containing only the -71T polymorphism. These data indicate that IFN-gamma suppresses native CYP2E1 promoter activity and that the -71G is critical for this response.

Pharmacogenomics (PG) holds promise for transforming medical therapeutics but the details of how the promise will become reality are still vague. In this article, we focus on the role that laboratory medicine, as a discipline, might play in transitioning the application of pharmacogenomics into the healthcare system and begin to frame a perspective on how PG may be viewed in this context. Development of clinical diagnostic tests usually evolves as a continuum of information starting with the discovery of a potential biological marker through to its routine use in clinical practice. This process has traditionally been rooted in the practice of laboratory medicine and, importantly, includes the development of testing strategies to optimize the predictive value of single or a combination of biological markers. In this context, we also discuss a perspective on some future strategies that may prove useful in advancing the application of PG, including the need for an evidenced-based approach and the potential role of proteomics as a means to drive more comprehensive strategies.

Background: The dose response relationship of warfarin is unpredictable. Polymorphism of the Cytochrome P4502C9 enzyme leads to warfarin hypersensitivity presumably due to decreased metabolism of the S-enantiomer. The purpose of this study was to further characterize the relationship between CYP2C9 genotype and phenotype and to develop a basis for guidelines to interpret CYP2C9 genotype for warfarin dosing., Methods and Results: Patients stabilized on warfarin therapy were recruited from an anticoagulation clinic. Patients were genotyped for CYP2C9*2, CYP2C9*3 and CYP2C9*5 alleles by standard methods of polymerase chain reaction amplification and restriction endonuclease digestion. Phenotype was determined by; dose (mg/kg/d) required to maintain anticoagulation, (INR 2.0-3.0), oral plasma S-warfarin clearance, and the plasma S:R-warfarin ratio. In this cohort, no subjects were found to have the CYP2C9*5 allele. The plasma S-warfarin concentration did not differ with age, dose or CYP2C9 genotype. Both CYP2C9*2 and *3 alleles were associated with lower maintenance dosages, lower total and R-warfarin plasma concentrations, decreased oral clearance of S-warfarin, increased plasma S:R-warfarin ratio and extended S-warfarin elimination half-life. Advancing age was found to decrease Warfarin maintenance dose in subjects with the common active CYP2C9*1/*1 genotype but did not influence dose requirement of subjects with one or more variant CYP2C9 alleles., Conclusions: Subjects who have been titrated to a consistent target INR demonstrate comparable plasma S-warfarin concentrations independent of CYP2C9 genotype. The warfarin dose required to maintain a consistent target INR between subjects differs as a function of S-warfarin clearance which is decreased by both CYP2C9*2 and or CYP2C9*3 variant alleles. The variables of CYP2C9 genotype and age can be applied to restrict the dosage range considered for individual patients.