Comparability of a new turbidimetric digoxin test with other immunochemical tests and with HPLC—a multicenter evaluation

A new turbidimetric inhibition immunoassay for digoxin (Tina-quant® □a Digoxin, Boehringer Mannheim) was evaluated in seven laboratories. It can be performed without sample pretreatment with ready-to-use reagents on nondedicated analyzers in combination with routine clinical chemistry. The studies revealed a good analytical performance: lower limit of detection 0.12 μg/L (3 SD from mean of blank); linearity up to 7.5 μg/L; median between-run CVs 8.1% (0.6 μg/L), 2.8% (1.5 μg/L), 1.9% (3 μg/L); mean analytical recovery in control sera 98–102%; slopes from 0.97 to 1.09 and intercepts from −0.28 to 0.10 μg/L in comparison with four immunoassays; and a high resistance to common interferents. The test was more resistant to digoxin-like immunoreactive factor (DLIF) interference than other methods, showing cross-reactivity only in some intensive care patient samples. Among 192 patients in whom DLIF is expected (e.g., pregnant women, patients with renal failure, newborns), 90% of results were ≤0.26 μg/L digoxin. Cortisol showed no cross-reactivity and digoxigenin had a low reactivity. An interlaboratory survey revealed a good comparability of the Tina-quant □a test with the median of all methods (slope 0.99, intercept −0.06 μg/L). An HPLC method for digoxin based on isocratic separation of samples on an RP-18 column followed by detection by an immunoassay yielded a reasonable comparability with the immunochemical tests with noncritical samples. Divergent results of immunoassays caused by DLIFs or different cross-reactivities with digoxin metabolites or derivatives can be explained by the use of this HPLC method.

Effect of antibody specificity on results of selected digoxin immunoassays among various clinical groups

We examined the specificity of three automated digoxin immunoassays (Abbott TDxFLx Digoxin II assay, Baxter-Dade Stratus II Digoxin assay, and Ciba Corning ACS Digoxin assay) applied without modification to (a) sera from 229 digoxin-free patients in 12 cohorts associated with nonspecific or endogenous digoxin-like immunoreactive factor (DLIF) interference, and (b) drug-free serum supplemented with the major metabolites and analogs of digoxin. We observed three patterns of apparent digoxin results among the DLIF samples: one common to kidney and liver failure patients, where TDx and Stratus assays showed significant positive results; one common to newborns and cord blood, where only the TDx assay had significant interference; and one from cardiac surgery patients, where the Stratus assay alone showed interference. Of the three assays, the ACS had the least interference from DLIF. The assays also behaved differently with respect to cross-reactivity with digoxin metabolites, digitoxin, and digitoxin metabolites. The ACS assay again had the least analog or metabolite cross-reactivity. The three methods agreed well on digoxin-positive specimens, with a mean bias of <0.15 microgram/L digoxin for each and discrepancies (defined as >3 SD between the assay pairs compared) of only 3-5%.

Digoxin Immune Fab Administration following an Unexplained Increase in Serum Digoxin Concentration

Objective: To report a case of digoxin immune Fab (DIF) administration following an unexplained increase in serum digoxin concentration in an asymptomatic patient with chronic renal failure. Case Summary: A 70-year-old man presented to the hospital with congestive heart failure, atrial fibrillation, chronic renal failure, and suspected digoxin toxicity. By day 3, he developed a more stable cardiac rhythm with nodal beats. His last known digoxin dose was 12 hours prior to admission. No explanation for an elevated serum digoxin concentration 48 hours after admission could be found. Despite absence of other signs of digoxin toxicity, DIF 80 mg iv was administered, and was immediately followed by 40 mg. Discussion: This case illustrates that elevated digoxin concentrations may be observed in patients with renal failure. These may not be true high concentrations because of the following potential factors: (1) the presence of digoxin-like factors, (2) increased biotransformation of digoxin, and (3) accumulation of metabolites that interfere with the assay. Digoxin metabolites are known to cross-react with the antibodies in commonly used digoxin immunoassays, and may be inappropriately interpreted to signal digoxin toxicity. Both the accuracy and reliability of digoxin immunoassay techniques have been questioned or challenged over the years. It is difficult to determine whether a reported toxic serum digoxin concentration represents the true concentration or cross-reactivity between digoxin metabolites and antibodies used in most digoxin immunoassays. Data Sources: Data collection sources included retrospective review of patient medical records, personal contact with one of the physicians involved in rendering patient care for interpretation of the electrocardiogram changes, clinical symptoms and rationale for DIF administration, and contact with the immunoassay technologist, who indicated that the fluorescence polarization immunoassay technique was used for analysis of digoxin concentrations. The medical literature then was reviewed. Conclusions: DIF should be reserved for use in symptomatic patients. Elevated digoxin concentrations must be evaluated for various factors that can cause falsely elevated values. Clinical signs and symptoms are critical in making the decision to use Fab. Antidotal measures should be based on correlation of patient symptoms with serum digoxin concentrations.

Digoxin immunoassay with cross-reactivity of digoxin metabolites proportional to their biological activity

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.

An update on digoxin.

This review deals briefly with recent developments in the therapeutic drug monitoring of digoxin. Strategies for decreasing the interference by digoxin metabolites, digoxin-like factors, and spironolactone metabolites in immunoassays of digoxin are discussed. Other issues addressed include the development of alternative methods of analysis, such as receptor assays and "high-pressure" liquid chromatography; digoxin-like factors in hypertension; drug-drug interactions; redistribution of digoxin stores in the body; and forensic considerations.