Positive and negative interference of dlis in serum digoxin measurement impact on tdm of digoxin

Positive interference of DLIS in the FPIA digoxin assay (Abbott Laboratories) is very well documented in literature. Many investigators used this assay to measure DLIS levels in various patients not receiving digoxin. Avendano et al. reported 89% false-positive digoxin values in blood drawn from peripheral veins of neonates and a striking 100% false-positive digoxin levels in the corresponding cord blood when FPIA (Digoxin II) was used for the measurement. The authors also observed 60% false-positive values in patients with severe hepatic disease and concluded that digoxin levels must be interpreted very carefully in these patients (37). Frisolone et al. studied apparent serum digoxin levels in patients with liver disease using FPIA (Digoxin II, TDx analyzer), RIA (Gamma goat I 125), and a fluorometric assay (Stratus, Dade). These patients did not receive digoxin or spironolactone. The authors observed measurable apparent digoxin concentrations in 57% of the patients using the RIA (range: 0.2-0.6ng/mL), 55% in patients using the FPIA (range: 0.2-1.56 ng/mL), and only 28% patients with the fluorometric assay (range: 0.2-0.38 ng/mL) and concluded that FPIA and RIA digoxin assays were more susceptible to DLIS interference (10). Datta et al. studied potential interference of DLIS with different digoxin assays and concluded that chemiluminescent assay (CLIA on ACS:180 analyzer, Bayer Diagnostics) and the fluoroimmunoassay (Stratus, Baxter Corporation) showed almost no interference from DLIS compared with an RIA (Magic, Ciba-Corning) (38).

Miller et al. studied analytical performance of the CLIA digoxin assay on the ACS:180 analyzer (Ciba-Corning currently marketed by Bayer Diagnostics) by comparing this assay with the FPIA (Digoxin II), Stratus II digoxin assay, and an RIA digoxin assay (Magic RIA, Ciba-Corning). The authors detected no DLIS in sera using CLIA, but measurable concentrations of DLIS were observed with the FPIA, Stratus II digoxin assay, and the RIA method. The authors also compared digoxin levels in 121 sera from 49 patients and observed comparable values with all digoxin assays. However, 11 patients showed discrepant digoxin values (>2Sy x from the regression line). These patients had renal disease or hepatic disease. The discrepant digoxin values were always lower with the CLIA compared with other digoxin assays. The authors concluded that the CLIA digoxin assay on the ACS:180 analyzer had improved specificity for digoxin (30).

Way et al. evaluated Vitros digoxin assay (Johnson and Johnson) for interference from DLIS. The Vitros digoxin assay is an enzymatic heterogeneous competitive immunoassay that uses dry slide technology. The authors compared this assay with the Online digoxin assay (Roche), which is a homogenous microparticle immunoassay based on the aggregation of digoxin-coated microparticles in the presence of anti-digoxin antibody. Digoxin in the specimen partly inhibits aggregation, and thus, the rate of aggregation (as measured by light scattering) is inversely proportional to digoxin concentration. The authors also used an MEIA (Abbott Laboratories) that utilizes digoxin-alkaline phosphatase conjugate and 4-methyllumbelliferyl as a substrate. The authors compared three digoxin assays using 26 adult patients receiving digoxin and observed mean digoxin concentrations of 1.30 ± 0.69 ng/mL (SD) by Roche assay, 1.34 ± 0.58 ng/mL by the Abbott assay, and 1.46 ± 0.68 ng/mL by the Vitros digoxin assay. To study the potential interference of DLIS (suspected cause of such discrepancy) in these assays, the authors added known amounts of digoxin to serum samples prepared from newborns (high DLIS) and adults (no DLIS). The samples from newborns showed a mean digoxin level of 0.41 ng/mL by the Roche method and 0.7 ng/mL by the Vitros. The specimens from adults showed a mean value of 0.7 ng/mL by the Roche method and 0.8 ng/mL by the Vitros. The authors concluded that the positive bias in the Vitros assay compared with Roche OnLine assay was probably because of DLIS (39).

Bonagura et al. (40) reported high specificity of the Roche OnLine assay for digoxin, which had no cross-reactivity with DLIS and negligible cross-reactivity with noncar-dioactive metabolites of digoxin. Marzullo et al. (41) reported that the EMIT 2000 digoxin immunoassay and the Roche OnLine digoxin immunoassay were least affected by DLIS compared with other digoxin assays. Saccoia et al. also confirmed improved specificity of the EMIT 2000 digoxin assay and very low cross-reactivity from DLIS compared with the FPIA digoxin assay and concluded that the EMIT 2000 had adequate specificity, sensitivity, precision, and accuracy for routine monitoring of digoxin in clinical laboratories (42). More recently marketed digoxin immunoassays such as a turbidimetric assay on ADVIA 1650 analyzer and an enzyme-linked immunosorbent digoxin assay on the ADVIA IMS 800 I system (both marketed by Bayer diagnostics) are virtually free from DLIS interference (43,44). This may be related to the use of specific monoclonal antibodies targeted against digoxin in this new assay compared with rabbit polyclonal antibody targeted against digoxin in the FPIA.

Although most investigators reported positive interference of DLIS with serum digoxin measurement, negative interference (falsely lower digoxin values) of DLIS in the MEIA for digoxin has been reported (45). This may result in digoxin toxicity because a clinician may increase a digoxin dose based on a falsely low digoxin concentration because of elevated DLIS (46).

3.1. Elimination of DLIS Interference in Digoxin Immunoassay by Ultrafiltration

Although several monoclonal antibody-based digoxin immunoassays are virtually free from DLIS interference, ultrafiltration technique can be used to completely eliminate DLIS interference in serum digoxin measurement by immunoassays. Valdes and Graves (47) reported strong serum protein binding of DLIS. Therefore, DLIS is usually absent in the protein-free ultrafiltrate. Taking advantage of high protein binding of DLIS and poor protein binding of digoxin (25%), both positive and negative interference of DLIS in serum digoxin measurement, can be completely eliminated by measuring digoxin concentration in the protein-free ultrafiltrate (48,49). Protein-free ultrafiltrate of digoxin can be easily prepared by centrifuging the specimen at 1500-2000 xg in a Centrifree Micropartition System (Ultrafiltration device, Amicon, distributed by Millipore Corporation; Molecular weight cutoff 30,000 Da) for 20-30 min at room temperature. Digoxin is only 25% protein bound and approximately 75% of digoxin is found in the ultrafiltrate. Immunoassay kits used for monitoring total digoxin concentration have adequate sensitivity to measure free digoxin concentration in the protein-free ultrafiltrate. It is also possible to calculate true total digoxin concentration and the extent of DLIS interference in digoxin measurement by measuring albumin, total and free digoxin concentrations, and then using mathematical equations (50).

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