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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.

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.

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.

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: 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.

Background: Ouabain-like factor (OLF) and its newly discovered reduced species, dihydroouabain-like factor (Dh-OLF), are mammalian cardenolides whose structural and functional characteristics are similar to the plant-derived compounds ouabain and dihydroouabain. These endogenous compounds are believed to be produced by the adrenals and to constitute part of an hormonal axis that may regulate the catalytic activity of the alpha-subunit of Na(+),K(+)-ATPase. We developed antibodies sufficiently specific to distinguish between OLF and Dh-OLF, and in this study demonstrate the selective secretion of OLF and Dh-OLF from human H295R-1 adrenocortical cells in culture., Methods: We used reversed-phase HPLC, inhibition of Na(+),K(+)-ATPase catalytic activity, and two enzyme immunoassays developed with antibodies specific to ouabain and dihydroouabain to purify and characterize the secretion of these two compounds by human adrenal cells in culture. Purified antisera had high titers (1 x 10(6) for ouabain and 8 x10(5) for dihydroouabain) and were specific to their corresponding antigens., Results: Human H295R-1 cells grown in serum-free medium secreted 0.18 +/- 0.03 pmol of OLF and 0.39 +/- 0.04 pmol of Dh-OLF per 10(6) cells in 24 h. Both OLF and Dh-OLF inhibited the ouabain-sensitive catalytic activity of the sodium pump (0.03 micro mol/L OLF inhibited 29% of the catalytic activity; 0.07 micro mol/L Dh-OLF inhibited 17%). Stimulation of the cell culture by dibutryl cAMP increased the secretion of Dh-OLF 50% over control (unstimulated), whereas the secretion of OLF did not increase significantly., Conclusions: OLF and Dh-OLF are secreted by human adrenal cells, and antibodies specific to these two compounds can be developed, using the plant-derived counterparts as antigens. The secretion of Dh-OLF is responsive to a cAMP-dependent stimulation mechanism, whereas OLF is not. Our data suggest that either the secretory or biosynthetic pathways for production of these two compounds by human adrenal cells may have different control mechanisms or that they may be linked via a precursor-product relationship.

Background: Domestic and international acts of terrorism using chemicals and pathogens as weapons have recently attracted much attention because of several hoaxes and real incidents. Clinical laboratories, especially those affiliated with major trauma centers, should be prepared to respond rapidly by providing diagnostic tests for the detection and identification of specific agents, so that specific therapy and victim management can be initiated in a timely manner. As first-line responders, clinical laboratory personnel should become familiar with the various chemical or biological agents and be active participants in their local defense programs., Approach: We review the selected agents previously considered or used in chemical and biological warfare, outline their poisonous and pathogenic effects, describe techniques used in their identification, address some of the logistical and technical difficulties in maintaining such tests in clinical laboratories, and comment on some of the analytical issues, such as specimen handling and personal protective equipment., Content: The chemical agents discussed include nerve, blistering, and pulmonary agents and cyanides. Biological agents, including anthrax and smallpox, are also discussed as examples for organisms with potential use in bioterrorism. Available therapies for each agent are outlined to assist clinical laboratory personnel in making intelligent decisions regarding implementation of diagnostic tests as a part of a comprehensive defense program., Summary: As the civilian medical community prepares for biological and chemical terrorist attacks, improvement in the capabilities of clinical laboratories is essential in supporting counterterrorism programs designed to respond to such attacks. Accurate assessment of resources in clinical laboratories is important because it will provide local authorities with an alternative resource for immediate diagnostic analysis. It is, therefore, recommended that clinical laboratories identify their current resources and the extent of support they can provide, and inform the authorities of their state of readiness.

Background: Xenotransplantation of pig hearts to humans could be hampered by the reportedly reduced affinity for digoxin of pig heart. We examined the hypothesis that expression of the individual alpha-subunit isoforms of the sodium pump [Na(+),K(+)-ATPase (NKA)], the receptor for the plant-derived cardiac glycosides, may be responsible for this difference., Methods: We used a NKA-inhibition assay in combination with Western analysis, immunohistochemistry, and phosphorylation of the NKA alpha subunit to identify the distribution and expression of alpha isoforms in four chambers of porcine and human hearts., Results: We confirmed that tissue from porcine heart is less sensitive to digitalis (IC(50) = 1740 nmol/L) when compared with human heart (IC(50) = 840 nmol/L), whereas porcine cerebral cortex-mix had an affinity comparable to that of human heart (IC(50) = 910 nmol/L). Our data show that porcine cerebral cortex-mix and human heart contain all three alpha isoforms, whereas porcine heart expresses only the alpha1 isoform., Conclusions: The different expressions of sodium pump isoforms in human vs porcine cardiac tissues suggests that porcine hearts may not be pharmacologically or endocrinologically compatible when used in humans. Studies of both pharmacologic and endocrinologic tissue compatibility are needed prior to selection of organs for xenotransplantation.

The Sixth Conference on the "Standards of Laboratory Practice Series", sponsored by the National Academy of Clinical Biochemistry (NACB), was held on August 4-5, 1998, at the Annual Meeting of the American Association for Clinical Chemistry, in Chicago, IL. An expert committee was assembled to write recommendations on the use of cardiac markers in coronary artery diseases. The NACB Committee prepared a preliminary draft of the guidelines, made them available on the World Wide Web (www.nacb.org), and distributed them before the presentations. The recommendations were divided into four areas: the use of markers in the triage of patients with chest pain, acute coronary syndromes, clinical applications other than acute myocardial infarction and research, and assay platforms and markers of acute myocardial infarction. The recommendations were revised and subsequently re-presented in part at the "Biomarkers in Acute Cardiac Syndromes Conference", sponsored by the Jewish Hospital Heart and Lung Institute, Louisville KY, on October 16-17, 1998. This report lists each recommendation, its scientific justification, and a summary of discussions from conference participants and reviewers. Approximately 100 individuals responded to various versions of these recommendations via direct correspondences, telephone calls to Committee members, electronic mail correspondence to the Committee Chairman, or oral questions and comments raised during one of the two conference presentations. Some of the recommendations were changed to reflect the consensus opinion. In cases in which there was no consensus, the Committee included pertinent discussion without necessarily changing the original recommendations. At times, the Committee members felt that although a particular recommendation might not be the current standard of care today, they anticipate that it likely will be adopted in the near future.

This multicenter study evaluated the Biosite Triage(R) Cardiac Panel as a quantitative, multimarker, whole blood system for the detection of acute myocardial infarction (MI). Optimum cutoffs for the discrimination of acute MI (n = 192 patients, 59 with MI) as determined by ROC curve analyses were as follows: 0.4 microgram/L for cardiac troponin I (cTnI); 4.3 microgram/L for the creatine kinase MB isoenzyme (CK-MB); and 107 microgram/L for myoglobin. The Triage Panel showed the following concordances for detection or rule-out of MI compared with established devices: cTnI >89%; CK-MB >81%; myoglobin >69%. No significant differences were present between methods for the same marker. Diagnostic efficiencies demonstrated comparable sensitivities and specificities for the diagnosis of MI in patients presenting with symptoms compared with the Dade, Beckman, and Behring CK-MB, cTnI, and myoglobin assays; the ratio of sensitivity to specificity for each marker was as follows: cTnI, 98%:100%; CK-MB, 95%:91%; myoglobin, 81%:92%. The areas under the ROC curves for the Biosite myoglobin, CK-MB, and cTnI were 0.818, 0.905, and 0.970, respectively; the areas were significantly different, P <0.05. In patients with skeletal muscle injury and renal disease, the Triage cTnI showed 94% and 100% specificity, respectively. The Triage panel offers clinicians a whole blood, point-of-care analysis of multiple cardiac markers that provides excellent clinical sensitivity and specificity for the detection of acute MI.

In this Standard of Laboratory Practice we recommend guidelines for therapeutic monitoring of cardiac drugs. Cardiac drugs are primarily used for treatment of angina, arrhythmias, and congestive heart failure. Digoxin, used in congestive heart failure, is widely prescribed and therapeutically monitored. Monitoring and use of antiarrhythmics such as disopyramide and lidocaine have been steadily declining. Immunoassay techniques are currently the most popular methods for measuring cardiac drugs. Several reasons make measurement of cardiac drugs in serum important: their narrow therapeutic index, similarity in clinical complications and presentation of under- and overmedicated patients, need for dosage adjustments, and confirmation of patient compliance. Monitoring may also be necessary in other circumstances, such as assessment of acetylator phenotypes. We present recommendations for measuring digoxin, quinidine, procainamide (and N-acetylprocainamide), lidocaine, and flecainide. We discuss guidelines for measuring unbound digoxin in the presence of an antidote (Fab fragments), for characterizing the impact of digoxin-like immunoreactive factor (DLIF) and other cross-reactants on immunoassays, and for moni-toring the unbound (free fraction) of drugs that bind to alpha1-acid glycoprotein. We also discuss logistic, clinical, hospital, and laboratory practice guidelines needed for implementation of a successful therapeutic drug monitoring service for cardiac drugs.

Pharmacogenetics is the study of the linkage between an individual's genotype and that individual's ability to metabolize a foreign compound. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Phenotypes exhibiting poor and ultraextensive metabolism result from genetic variance (polymorphism) of enzymes involved in metabolism. Thus, in pharmacogenetic studies one applies genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug metabolism phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic efficiency. More than 25 commonly prescribed medicines are metabolized by the cytochrome P-4502D6 (CYP2D6) isoenzyme, and polymorphism of the CYP2D6 gene affects the therapeutic management of up to 17% of individuals in some ethnic groups. In this review, we summarize and update information concerning drug-metabolizing genotypes with emphasis on CYP2D6 genotyping techniques that can be applied by the clinical laboratory for linking human genetics to therapeutic management.

Ingestion of oleander plant, containing the cardiac glycoside oleandrin, has been reported to induce fatal poisonings. Derivatives of oleandrin are structurally similar to digoxin. We investigated the cross-reactivities of oleandrin and its aglycone metabolite, oleandrigenin, in several commercially available digoxin immunoassays; assessed their ability to inhibit Na,K-ATPase catalytic activity; and measured their binding to proteins in serum. As assayed with ACS:180, Stratus, RIA, On-Line, and TDx digoxin assays, oleandrin at 100 micromol/L in digoxin-free serum gave apparent digoxin values of 0, 0.83, 2.24, 2.37, and 5.34 nmol/L, respectively, whereas oleandrigenin at that concentration gave results of 0, 0.52, 0.77, 4.94, and 1.40 nmol/L. Study of Na,K-ATPase inhibition showed IC50 values (micromol/L) of 0.22 for ouabain, 0.62 for oleandrin, 1.23 for oleandrigenin, and 2.69 for digoxin. At 25 degrees C, 96% of oleandrin and 48% of oleandrigenin were bound to serum proteins. Because detection of oleandrin and oleandrigenin by digoxin immunoassays is variable between assays as well as between congeners, assessment of cross-reactivity is warranted for each assay. The inhibition of Na,K-ATPase by oleandrin and oleandrigenin confirms that they likely exert their toxic effects through inhibition of sodium pump activity. In cases of digitalis-like poisoning with suspicion of oleander ingestion, a combination of digoxin immunoassays may be useful to effectively rule out the presence of oleander.

Digoxin-like immunoreactive factor (DLIF) from adrenal glands is an endogenous ligand structurally related to the plant-derived cardiac glycoside digoxin. Cardiac glycosides regulate the activity of the sodium pump and thus play key roles in disease processes involving regulation of ion transport. We now report the discovery of an endogenous dihydro-DLIF analogous to dihydrodigoxin. We used HPLC, ultraviolet spectrophotometry, and cross-reactivity with two antibodies, one specific for digoxin and one for dihydrodigoxin, to support the hypothesis that dihydro-DLIF contains a chemically reduced lactone ring. The spectral absorbance maximum for dihydro-DLIF is at 196 nm, identical to dihydrodigoxin. DLIF and dihydro-DLIF are 975- and 2588-fold less immunoreactive than digoxin and dihydrodigoxin for their respective antibodies. The molar ratio of dihydro-DLIF to DLIF is approximately 5.3 in bovine adrenocortical tissue and approximately 0.38 in human serum. Dihydrodigoxin (reduced lactone ring) added to microsomes isolated from bovine adrenal cortex produced a 4.5-fold increase in digoxin-like immunoreactivity (oxidized lactone ring) after 3 h of incubation. The biotransformation is likely mediated by a cytochrome P-450 NADPH-dependent process. Our findings demonstrate the presence of a dihydro-DLIF in mammals and suggest a metabolic route for synthesis of endogenous DLIF in mammalian tissue.

Our objective was to identify commercially available digoxin immunoassays whose cross-reactivity with digoxin metabolites paralleled the pharmacological activity of the metabolites. We measured the immunoreactivity of digoxigenin bis- and monodigitoxosides, digoxigenin, and dihydrodigoxin in four immunoassays and compared the immunoactivities with pharmacological activities from studies involving whole-animal and receptor (Na,K-ATPase)-based assays. Correlation coefficients for comparisons of immunoassay reactivity and human heart receptor reactivities were: ACS, 0.96; TDx, 0.60; Stratus, 0.57; and Magic, 0.42. Comparison with other biological assays showed a similar trend. The major difference in metabolite cross-reactivities among the immunoassays was that of digoxigenin (ACS, 0.7%; TDx, 103%; Stratus, 108%; Magic, 153%), which has approximately 10% bioactivity relative to digoxin. Measured recovery of mixtures of digoxin and metabolites confirmed these findings. We conclude that the monoclonal antibody in the ACS digoxin assay closely mimics Na,K-ATPase in detecting digoxin and its metabolites. This finding provides a basis for developing therapeutic drug monitoring immunoassays capable of approximating the true pharmacological activity of a mixture of drug metabolites.

Na,K-ATPase (sodium pump; EC 3.6.1.37) is present in the membrane of most eukaryotic cells and controls directly or indirectly many essential cellular functions. Regulation of this enzyme (ion transporter) and its individual isoforms is believed to play a key role in the etiology of some pathological processes. The sodium pump is the only known receptor for the cardiac glycosides. However, endogenous ligands structurally similar to digoxin or ouabain may control the activity of this important molecular complex. Here we review the structure and function of Na,K-ATPase, its expression and distribution in tissues, and its interaction with known ligands such as the cardiac glycosides and other suspected endogenous regulators. Also reviewed are various disorders, including cardiovascular, neurological, renal, and metabolic diseases, purported to involve dysfunction of Na,K-ATPase activity. The escalation in knowledge at the molecular level concerning sodium pump function foreshadows application of this knowledge in the clinical laboratory to identify individuals at risk for Na,K-ATPase-associated diseases.

The presence of salicylates in urine reduces the signal in Emit assays (Syva), potentially yielding false-negative drugs-of-abuse screening results. We demonstrate that the principal urinary metabolite of salicylate, salicyluric acid (SUA; 2-hydroxybenzoylaminoacetic acid), interferes with the measurement of NADH formed in the assay by reducing the molar absorptivity of NADH at 340 nm. Thus, for a given concentration of d.a.u. analyte the change in absorbance over the assay time interval is less in the presence of SUA. With the Emit cocaine assay on the Hitachi 704 analyzer, the rate of absorbance change (delta AR) monitored at 340 nm for a specimen containing approximately 270 micrograms/L benzoylecgonine (BE) was 57 +/- 1.9 mA/min without SUA and 29 +/- 2.7 mA/min with 5 g/L SUA (n = 20). In contrast, delta AR determined at 376 nm was 18.6 +/- 0.5 mA/min with and 17.9 +/- 0.8 mA/min without 5 g/L SUA (n = 20). Measuring the Emit assay signal at wavelengths where SUA has no absorbance (376 nm) eliminates the interference due to SUA while maintaining the precision of the assay near the cutoff concentration for BE (300 micrograms/L).

We evaluated the analytical and clinical performance of an immunoassay for cardiac troponin T (cTnT). Within-run and total imprecision ranged from 1.6 to 11.3%. The sensitivity and linear range was 0.015 and 13 micrograms/L, respectively. Frozen samples were stable for at least 8 weeks. No interferences were seen with lipids or bilirubin (total and conjugated). Hemoglobin caused a negative bias at concentrations > 4 g/L. Heparinized plasma showed a 6% negative bias compared with serum. The clinical utility of cTnT was compared with that of creatine kinase (CK)-MB (mass assay). The sensitivity of cTnT measurements from 63 patients with acute myocardial infarction (AMI) (cTnT cutoff 0.1 microgram/L) was 60% at 0-3 h, 59% at 3-6 h, 94% at 6-9 h, 90% at 9-12 h, 99% at 12-24 h, 92% at 24-48 h, 83% at 48-72 h, and 100% at 72-96 h. Corresponding results for CK-MB (cutoff 5.0 micrograms/L and 2.5% relative index) were 45%, 64%, 82%, 97%, 87%, 81%, 54%, and 59%. The specificity of the markers from 49 non-AMI patients was 46% and 79% for cTnT and CK-MB, respectively. We show that CK-MB is more specific for diagnosis of AMI, and propose that cTnT is more sensitive to myocardial injury.

During routine drug analysis with the Syva d.a.u. Emit immunoassays we observed a high frequency of urines with lower rates of changes in absorbance (delta A R) than the rate for a drug-free urine calibrator. Many of these urines contained salicylates. Among 40 urines with apparent salicylate concentrations between 15 and 420 mg/dL tested for benzoylecgonine (BE), 20 had delta A R < -4 (range +2 to -28 mA/min). The rates decreased with increasing salicylate: delta A R = -0.057 x (salicylate, mg/dL) -0.22 mA/min (r = 0.85, n = 40, P < 0.01). Urines from 100 control subjects (no salicylate) had mean +/- SD delta A R values of -1.05 +/- 2.2 mA/min (range +3 to -7; only two were < -4 mA/min). Although direct addition of salicylic acid (200 mg/dL) to urine specimens did not reproduce the negative bias, ingestion of aspirin (acetylsalicylic acid) did by -0.09 mA/min per 1 mg/dL (72.4 mumol/L) salicylate. Negative biases observed for other Emit d.a.u. assays after salicylate ingestion lead us to conclude that ingestion of therapeutic doses of aspirin may cause false-negative results for drug screens in urines by this technology.

We evaluated the Clinistat Analyzer (Miles Inc., Diagnostics Division, Elkhart, IN) for measuring cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol at three medical centers. The system, based on multilayer film technology, uses precalibrated, dry film reagent disks. Ten microliters of serum is applied to the dry film reagent disk in the test procedure. For HDL-cholesterol measurement, serum is pretreated by precipitation with phosphotungstic acid and magnesium chloride. Total precision (CVs) of each of the three assays was less than or equal to 5%. The assay ranges were linear and satisfactory for clinical use. Patients' results compared well with established methods. No significant interferences were found with hemolysis, icterus, and lipemia.

Here we review techniques useful in eliminating or reducing interferences caused by molecules that cross-react in immunoassays. The biochemical rationale for using these techniques is discussed. Examples are taken from recent studies aimed at reducing interferences caused by endogenous molecules such as digoxin-like immunoreactive factors or steroid hormones. In this context the role of protein-binding of cross-reacting molecules is also considered. Immunoassay ligand selectivity can be inherently limited by the heterogeneity of the antigenic response or by the structural similarity of epitopes on multiple ligands. Certain empirical approaches have proved useful in maximizing the analytical specificity of immunoassays. These approaches include isolating the relevant ligands before immunoassay, adjusting the kinetic or equilibrium conditions used during the assays, and developing more specific antisera. The physicochemical properties of the cross-reacting molecule best dictate which technique(s) to use. The approaches discussed here are general and apply to minimizing interference caused by a wide variety of both endogenous and exogenous cross-reacting molecules.

We report an evaluation of the GammafloTM automated continuous-flow radioimmunoassay instrument in which we used a digoxin assay to assess system performance. System operation was based on combined continuous-flow and column-chromatographic techniques. No drift or carryover was detectable in 180 within-assay consecutive determinations performed at a rate of 42 determinations per hour (5 h of continuous operation). Within-assay and between-assay precision were less than 6% (coefficient of variation). The automated method correlated well (r = 0.960 and 0.952, respectively) with two established manual digoxin radioimmunoassay procedures. The data suggest this automated system offers a valid alternative to manual radioimmunoassay procedures in terms of overall precision, simplicity of operation, and sample throughout capacity.

We present guidelines for assessing the performance of nonquantitative (qualitative) assay methods. Criteria to be evaluated include analytical sensitivity, imprecision near limits of detection, analytical specificity, accuracy over a wide range of analyte concentrations, potential interferents, and technical ease of performance. A protocol was developed to evaluate several nonquantitative assay kits for detection of human choriogonadotropin (hCG) in serum. These include Tandem Icon HCG (Hybritech), Quest Pregnancy Test (Quidel), Concep-7 beta hCG (Leeco), and Beta Quik V (Pacific Biotech). Quantitative measurement of beta-hCG by RIA (Immophase beta hCG, Corning Medical) was used as the reference method. Results of this evaluation are discussed. The guidelines established and utilized in this report are adaptable to the evaluation of assay kits that measure other analytes by qualitative techniques.

We studied the effects of pretreating urine samples with tetrasodium EDTA (TEDTA) before measuring urinary oxalate with an enzymatic kit (Sigma). Mean analytical recovery of added oxalic acid was only 49% (SD +/- 13%) when the assay was performed as recommended by the manufacturer, but treating samples with TEDTA improved recoveries (96 +/- 10%). In 20 unselected 24-h urine samples assayed with and without TEDTA treatment, the mean oxalate concentrations were significantly (P less than 0.001) different: 15.6 +/- 8.7 and 12.2 +/- 7.9 mg/L, respectively. TEDTA-treated urine samples stored for 14 days at -20 degrees C lost 20% of their oxalate concentration. Use of TEDTA simplifies sample preparation by eliminating the alkalinizing step needed to dissolve EDTA or disodium EDTA.

Various laboratories have reported endogenous digoxin-like immunoreactive factor(s) (DLIF) in blood from patients in renal failure or liver failure, from newborn infants, and from third-trimester pregnant women. Similar immunoreactivity has been detected in amniotic fluid, in cord blood, and in urine and serum from normal subjects. The factor(s) giving rise to this immunoreactivity cross react with antibodies used in many currently available immunoassays for digoxin, sometimes causing apparent digoxin concentrations exceeding the therapeutic range obtained for exogenous digoxin, with consequent errors in measurement and in subsequent clinical interpretation of digoxin results. Here, I summarize findings in our laboratory and those of others. DLIF evidently exist in three states in serum: tightly protein-bound, weakly protein-bound, and unbound (free). In normal subjects, greater than 90% of the total DLIF in serum is tightly but reversibly bound to serum proteins and is not readily detectable by direct measurement of digoxin in serum with conventional immunoassays. However, there seems to be a redistribution of the more weakly bound and unbound components in patients with renal failure, pregnant women, and newborns. The increased values detected in these groups are ascribable to increased amounts of weakly bound and unbound DLIF rather than to increased total DLIF. Carrier proteins may play a prominent role in the transport of these factors in blood. I discuss the potential physiological and pharmacological implications of detecting endogenous immunoreactive factors that cross react with antibodies to drugs.

We describe two centrifugal analyzer methods for measuring total bilirubin in serum. Diazotized 2-chloroaniline-5-sulfonic acid is used in both. In the first procedure, ethylene glycol and methanol are used as an accelerator solvent; results correlate well with those by a Jendrassik and Grof method adapted to the centrifugal analyzer, but there is considerable hemolysis interference. In the second method, dimethyl sulfoxide is used with the ethylene glycol/methanol solvent and almost all hemolysis interference is eliminated. For either method, a 15-microliter sample is required. In the second method, instrument response is linearly related to concentration to 250 mg/L and the within-run precision (CV) is about 1%.

We evaluated and compared three different commercial kit immunoassays for alpha-fetoprotein (AFP) before we implemented our neural tube defect screening program. Each kit can be used with either serum or amniotic fluid. Analytical recovery ranges for AFP reference sera within each kit's standard curve limits (in kilo-int. units/L) were 97-108% (7.5-180) for the Kallestad kit, 77-101% (21.8-436) for Amersham, and 92-100% (0-177) for Hybritech. CVs, within each manufacturer's standard-curve limits, for combined intra-assay (amniotic fluid pools) and inter-assay (kit serum controls) averaged 3.6-7.3% (Kallestad), 2.4-9.3% (Amersham (y) kit results showed a correlation of r = 0.97, y = 1.05x + 5.5 kilo-int. units per liter of maternal serum (n = 66; range, 2.0-98.5). Gestational age did not influence these assay correlations. The Kallestad AFP assay demonstrated a maternal serum positivity rate of 2.9% at greater than or equal to 2.5 (n = 655) and 8.9% at less than 0.5 (n = 423) multiples of the median. All kits performed well analytically.

We describe a rapid enzyme immunoassay for carbamazepine with a centrifugal analyzer (Rotochem IIA-36). Reagent costs are reduced fourfold while good precision and sensitivity are maintained. Sample volume is 10 microliter, and as many as 28 patients' sera can be measured during an assay time of 225 s. Assay temperature is 30 degrees C, the wavelength 340 nn. Linearity is excellent for a carbamazepine concentration range of 1 to 12 mg/L; analytical recovery is quantitative. Results correlate well with those by liquid- and gas-liquid chromatography. Absorbance rates for each carbamazepine concentration are acquired by a multi-point kinetic rate program and a computer program provides a logit-log transformation of absorbance rate vs. concentration data for final calculations in the assay. Hemoglobin interference precludes analysis of severely hemolyzed specimens.

Elimination of endogenous digoxin-like immunoreactive factors (DLIF) that interfere with accurate measurement of digoxin requires use of a highly specific anti-digoxin antibody, or that DLIF be separated from digoxin before immunoassay. Several commercial digoxin-assay kits include a step for separating serum proteins and other substances from digoxin before immunoassay. We tested six different immunoassay methods (some having pretreatment steps) for their ability to detect DLIF in serum from patients in renal failure, pregnant women, and neonates, all of whom were not taking digoxin. Extracting digoxin on a column of derivatized silicagel eliminated detectable DLIF from serum as measured by enzyme immunoassay (EMIT; Syva Co.), but recovery of added digoxin was quantitative. In contrast, protein precipitation with 5-sulfosalicylic acid left significant amounts of DLIF in samples, most probably because the procedure (TDx assay; Abbott Labs.) disrupted protein-DLIF binding. A glass-bead radioimmunoassay (Immophase; Corning Medical) had the most digoxin-specific antisera. By preparative silica-gel-chromatography of serum we could eliminate or significantly minimize inaccurate digoxin measurements attributable to endogenous DLIF.

We evaluated four commercial kits for measuring serum ferritin, based on three techniques: immunoradiometric assay, radioimmunoassay, and enzyme immunoassay. The kits evaluated were those manufactured by Abbott Laboratories, Clinical Assays, Corning Medical, and Ramco. Two of the immunoradiometric kits showed satisfactory results with respect to sensitivity and precision; they should be useful in diagnosing individuals with uncomplicated iron-deficiency anemia. One of the immunoradiometric assay kits, however, showed a high-dose "hook effect," beginning at 10 mg of ferritin per liter. We modified this kit to eliminate this effect, at least to ferritin concentrations of 33 mg/L. (We observed a ferritin value as high as 47 mg/L in one patient.) Results with all these kits did not inter-compare well for ferritin concentrations greater than 300 micrograms/L, a finding that casts further doubt on the controversial use of serum ferritin measurement in cases of iron overload.

We studied the effect of varying water and salt intake on the renal excretion of endogenous digoxin-like immunoreactive factors (DLIF). DLIF were measured in human urine and serum by competitive displacement of 125I-labeled digoxin from anti-digoxin antibodies. Diuresis was selectively induced in normal healthy humans by acute water ingestion, and natriuresis was preferentially induced by acute saline ingestion. We found the amount of endogenous immunoreactivity excreted in urine to be correlated with urine flow rate but not with urinary sodium excretion. Urinary excretion of DLIF, normalized to creatinine, was 3.6-fold greater at a urine flow rate of 5.5 mL/min than at 0.5 mL/min. On the other hand, saline intake increased urine flow rate 1.9-fold and increased sodium excretion threefold, but did not affect urinary excretion of DLIF. Fractional excretion of DLIF was linearly related to fractional excretion of water. This study demonstrates that normalization of DLIF values to urinary creatinine does not make DLIF excretion independent of urine flow rate and underscores the need for information on urine flow rate when DLIF measurements in urine are being interpreted.

Use of immunoassays that do not detect endogenous digoxin-like immunoreactive factors (DLIF) in serum significantly improves the between-assay correlation of digoxin results for patients. We investigated five different immunoassay methods (Abbott, Clinical Assays, Corning, Du Pont, and Syva), measuring digoxin by all five assays in sera from 38 patients in renal failure and in 40 patients with normal renal function, all taking digoxin. The mean standard error of the estimate (Sy X x) of digoxin results (compared for all five assays) were significantly lower for patients with normal renal function than for patients in renal failure (0.148 vs 0.293 microgram/L, P less than 0.001). Assays previously shown (Clin Chem 1987;33:401) to be the least sensitive to DLIF (Syva and Corning) gave the lowest mean scatter about the regression (Sy X x = 0.192 microgram/L, renal failure; 0.114 microgram/L, normal renal function) for all 10 assay correlations. Evidently, discrepancies between digoxin values as measured by different immunoassay kits for patients with renal disease can be attributed to DLIF. Moreover, because inaccurate digoxin results attributed to DLIF may not be limited exclusively to groups of patients with known increased concentrations of DLIF, the possibility of "latent" DLIF interference may be a problem in many other human subjects.