Interference of endogenous digoxin-like immunoreactive factors in serum digoxin measurement is minimized in a new turbidimetric digoxin immunoassay on ADVIA 1650 analyzer

Endogenous digoxin-like immunoreactive factors (DLIF) cross-react with antidigoxin antibody and falsely elevate or lower measured serum digoxin concentrations, depending on the assay design. Recently, Bayer Diagnostics released a turbidimetric assay for digoxin on the ADVIA 1650 analyzer. We studied potential interference of DLIF with this new digoxin assay. We analyzed 40 serum specimens from patients who have pathologic conditions that may increase serum DLIF concentrations. These patients were never exposed to digoxin or other agents that may lead to a measurable digoxin concentration. We also analyzed five specimens from autopsy and five specimens from neonates. Apparent digoxin concentrations were measured using the new turbidimetric digoxin assay, the fluorescence polarization immunoassay (FPIA, Abbott Laboratories, Abbott Park, IL), and also the chemiluminescent immunoassay (CLIA, Bayer Diagnostics). We observed measurable apparent digoxin levels with the FPIA in 5 uremic patients (range 0.24-0.86 ng/mL), 6 patients with liver disease (range 0.21-0.72 ng/mL), in 3 patients in the third trimester of pregnancy (0.21-26 ng/mL), and in 3 neonates (range 0.21-0.46 ng/mL). Four out of 5 autopsy specimens showed measurable apparent digoxin concentrations (0.23-0.81 ng/mL). In contrast, only 1 specimen (a uremic patient) showed an apparent digoxin concentration of 0.26 ng/mL with the turbidimetric digoxin immunoassay (FPIA value 0.86 ng/mL, CLIA value 0.32 ng/mL). Because DLIF is absent in the protein-free ultrafiltrate, we also measured free digoxin concentrations in DLIF-positive patients to ensure that the apparent digoxin concentrations were caused by DLIF. We observed no apparent digoxin concentrations in the protein-free ultrafiltrate in any DLIF-positive specimens. When serum specimens containing elevated concentrations of DLIF but no digoxin were supplemented with a known concentration of digoxin, we observed falsely elevated digoxin concentrations by the FPIA, as expected. In contrast, we observed a good agreement between the target and observed concentrations when the new turbidimetric assay was used. We conclude that DLIF has minimal effect on serum digoxin measurements by the new turbidimetric assay.     

Rapid detection of oleander poisoning using digoxin immunoassays: comparison of five assays

Oleander is an ornamental shrub that grows in the United States, Australia, India, Sri Lanka, China, and other parts of the world. All parts of the plant are poisonous because the presence of cardiac glycoside oleandrin. Despite its toxicity, oleander extract is used in folk medicines. Because of its structural similarity, oleandrin cross-reacts with the fluorescence polarization immunoassay (FPIA) for digoxin. We studied the potential of detecting oleandrin in serum using 5 common digoxin immunoassays (FPIA, MEIA, both from Abbott; Beckman digoxin assay on Synchron LX, Chemiluminescent assay, CLIA from Bayer Diagnostics) and a recently FDA-approved turbidimetric assay on the ADVIA 1650 analyzer (Bayer). Aliquots of drug-free and digoxin-like immunoreactive substances (DLIS)-free serum pools were supplemented with ethanol extract of oleander leaves or oleandrin (Sigma Chemicals) in amounts expected in vivo after severe overdose. We observed significant apparent digoxin concentration with FPIA, Beckman, and the new turbidimetric assay (1 mL drug-free serum supplemented with 5.0 microL of oleander extract: apparent digoxin 2.36 ng/mL by the FPIA, 0.32 ng/mL by the MEIA, 0.93 ng/mL by the Beckman, 0.82 ng/mL by the new turbidimetric assay). The CLIA showed no cross-reactivity. Similar observations were made when serum pools were supplemented with oleandrin. Because cross reactivity should be tested in the presence of the primary analyte, we supplemented serum pools prepared from patients receiving digoxin with oleander extract or oleandrin. The measured digoxin concentrations were falsely elevated with the FPIA, Beckman, and turbidimetric assays, the highest false elevation being observed with the FPIA. Surprisingly, apparent digoxin concentrations were falsely lowered when MEIA was used. Digibind neutralizes free apparent digoxin concentration in vitro in serum pools supplemented with oleander extract, and this effect can be measured by the FPIA. We conclude that FPIA is most sensitive to detect the presence of oleander in serum. In contrast, the CLIA (no cross-reactivity) should be used for monitoring digoxin in a patient receiving digoxin and self-medicated with a herbal remedy containing oleander.     

Effect of Chinese medicines Chan Su and Danshen on EMIT 2000 and Randox digoxin immunoassays: wide variation in digoxin-like immunoreactivity and magnitude of interference in digoxin measurement by different brands of the same product

Chan Su is a Chinese medicine prepared from the skin gland of a Chinese toad and is used in treating arrhythmia and other heart diseases. Danshen is prepared from the Chinese medicinal plant and is used for various cardiovascular diseases including angina pectoris. The authors studied the potential interference of such medicines with the widely used EMIT 2000 (Dade Behring; Deerpark, IL) digoxin assay and the recently marketed Randox digoxin assay (Randox Laboratories Ltd, Antrim, United Kingdom) (both run on the Bayer ADVIA 1650 analyzer) (Bayer Diagnostics, Tarrytown, NY) and compared their results with an FPIA (Abbott Laboratories) and a chemiluminescent immunoassay (CLIA; Bayer Diagnostics) for digoxin. Aliquots of drug-free serum were supplemented with 1 microL ethyl acetate extract of Danshen or aqueous extract of Chan Su, and apparent digoxin concentrations were measured by all four digoxin immunoassays (FPIA, EMIT, Randox, CLIA). The authors also supplemented aliquots of several different serum pools prepared from patients taking digoxin with very small amounts of Chan Su or Danshen extract and compared digoxin values with the control digoxin values (serum pool containing no Chinese medicine). The authors observed no interference of Danshen in either EMIT, Randox, or CLIA assay but observed an interference with the FPIA assay. On the other hand, the authors observed high interference of Chan Su in the FPIA assay but moderate interference with the EMIT 2000 and Randox digoxin assays. CLIA assay was again free from any interference. The authors also observed a wide variation in digoxin-like immunoreactivity and magnitude of interference in digoxin immunoassay in different brands of Chan Su and Danshen, indicating poor quality control in manufacturing of these Chinese medicines. Taking advantage of the high protein binding of digoxin-like immunoreactive components of Chan Su, the authors further demonstrated that interference of Chan Su in EMIT 2000 and Randox assays can be mostly eliminated by monitoring free digoxin.     

Analytic performance evaluation of a new turbidimetric immunoassay for carbamazepine on the ADVIA 1650 analyzer: effect of carbamazepine 10,11-epoxide

Carbamazepine, an anticonvulsant, requires therapeutic drug monitoring. Recently Bayer HealthCare, Diagnostics Division released a turbidimetric immunoassay of carbamazepine on the ADVIA 1650 analyzer. We evaluated the analytic performance of this assay by comparing values obtained with this new assay in sera of 54 patients receiving carbamazepine with the values obtained by using a widely used fluorescence polarization immunoassay (FPIA) and a chemiluminescent immunoassay (CLIA). The new turbidimetric immunoassay for carbamazepine showed excellent precision. The low control showed a total CV of 4.9% (mean 2.86, SD 0.14 microg/mL), the medium control demonstrated a total CV of 3.5% (mean 7.79, SD 0.27 microg/mL), and the high control showed a total CV of 4.8% (mean 16.15, SD 0.78 microg/mL). The assay was linear up to a carbamazepine concentration of 20 microg/mL. The assay showed excellent dilution recovery and recovery of samples supplemented with carbamazepine (mean recovery 102.2%). We observed an excellent correlation between the values obtained by the FPIA (x-axis) assay and the new turbidimetric (y-axis) assay (y = 0.96 x - 0.46, r = 0.99, n = 54). We also observed excellent correlation between the values obtained by the CLIA (x-axis) and the turbidimetric (y-axis) assay (y = 1.10 x -0.32, r = 0.99, n = 54). However, the slope of 1.10 was higher than the slope of 0.96 observed with the regression equation obtained by using values obtained by the FPIA and the turbidimetric assay. The positive bias obtained with the new turbidimetric assay compared with the CLIA assay resulted from lower cross reactivity of carbamazepine 10,11-epoxide, the active metabolite of carbamazepine, with CLIA. On the other hand, the cross reactivity of the metabolite is similar between the new turbidimetric assay and the FPIA assay. We conclude that the new turbidimetric assay can be used for routine monitoring of carbamazepine in clinical laboratories.     

A new turbidometric digoxin immunoassay on the ADVIA 1650 analyzer is free from interference by spironolactone,potassium canrenoate,and their common metabolite canrenone

Spironolactone and potassium canrenoate (aldosterone antagonist diuretics) are often used with digoxin in clinical practice. It has been well documented in the literature that spironolactone, potassium canrenoate, and their common metabolite canrenone cross-react with the fluorescence polarization immunoassay (FPIA) for digoxin and falsely elevate measured serum digoxin concentrations. Recently a new turbidometric assay for digoxin became commercially available from Bayer Diagnostic for application on the ADVIA 1650 Chemistry analyzer. We studied the potential interference of these compounds in this new digoxin assay. Aliquots of drug-free serum were supplemented with therapeutic and above-therapeutic concentrations of spironolactone, canrenone, and potassium canrenoate, and apparent digoxin concentrations were measured. We observed apparent digoxin concentrations with the FPIA digoxin assay as expected but observed no apparent digoxin levels with the new turbidometric immunoassay. When serum pools prepared from patients receiving digoxin were supplemented with these compounds in concentrations expected in serum in patients receiving these medications, we observed falsely elevated digoxin levels with the FPIA digoxin assay, but no statistically significant change was observed with the new turbidometric assay. We conclude that the new turbidometric assay for digoxin is free from interference by spironolactone, potassium canrenoate, and their common metabolite canrenone.     

The new enzyme-linked immunosorbent digoxin assay on the ADVIA Integrated Modular System is virtually free from oleander interference

Despite known toxicity of oleander, this product is used in herbal preparations. Oleander interferes with various digoxin immunoassays. It is possible that a person taking digoxin also may take oleander-containing herbal products, and digoxin immunoassays interfering with oleander cannot be used for therapeutic monitoring of digoxin. Recently, Bayer Diagnostics introduced a new enzyme-linked chemiluminescent immunosorbent digoxin assay for application on the ADVIA IMS System (ECLIA-digoxin). We studied potential interference of oleander with this new digoxin assay and found that this assay is virtually free from oleander interference. When aliquots of drug-free serum pools were supplemented with ethyl alcohol extract of oleander leaf or pure oleandrin standard, we observed significant apparent digoxin concentration when measured by the fluorescence polarization immunoassay (FPIA) but minimal digoxin-like immunoreactivity using the ECLIA digoxin assay. Because cross-reactivity should be studied in the presence of primary analyte, we prepared 2 serum pools using sera from patients receiving digoxin. Then aliquots of first digoxin pool were supplemented with oleandrin standard and aliquots of second digoxin pool with oleander extract. We observed significant increases in apparent digoxin concentration in the presence of both oleandrin and oleander extract using the FPIA. However, we observed no statistically significant change in digoxin concentration when ECLIA digoxin assay was used, indicating that this assay is virtually free from oleander interference.     

Measuring endogenous digoxin-like substance and exogenous digoxin in the serum of low-birth-weight infants

The interference with three serum digoxin assay methods of endogenous digoxin-like substance (EDLS) in the serum of low-birth-weight (LBW) infants was assessed. The serum from 5-mL blood samples obtained from each of 19 LBW infants was divided into four 0.5-mL portions. Each portion was spiked with 10 microL of a distilled water-ethanol solution with or without digoxin to produce final digoxin concentrations of 0 (control), 0.49, 0.98, or 1.96 ng/mL. Each portion in each patient was then analyzed by radioimmunoassay (RIA), fluorescence polarization immunoassay (FPIA), and radial partition immunoassay (RPIA) using the control portions to measure EDLS. Serum digoxin concentrations measured by each assay method were calculated by subtracting the EDLS concentrations in the control portions from the measured digoxin concentrations in the spiked samples. The mean +/- S.D. concentrations of EDLS measured by RIA and FPIA were 0.26 +/- 0.13 ng/mL and 0.33 +/- 0.16 ng/mL, respectively. Of the 19 control samples assayed by RPIA, 18 had EDLS concentrations less than 0.1 ng/mL; one sample reflected an apparent concentration of 0.11 ng/mL. Mean recovered digoxin concentrations by RIA at each spiked digoxin concentration were significantly different from those obtained by FPIA and RPIA. A low but significant correlation was noted between EDLS concentrations in serum samples assayed by RIA and FPIA. The RPIA method appears to be preferred over the RIA and FPIA methods used in this study for serum digoxin analysis in LBW infants because of acceptable accuracy and minimal interference by EDLS.     

Effect of Chinese medicines Chan Su and Lu-Shen-Wan on serum digoxin measurement by Digoxin III, a new digoxin immunoassay

Chan Su and Lu-Shen-Wan are Chinese medicines that crossreact with digoxin immunoassays. Recently, Abbott Laboratories released a new digoxin immunoassay, Digoxin III. We studied potential interference of Chan Su and Lu-Shen-Wan with the Digoxin III assay by comparing results obtained by using Digoxin II and fluorescence polarization immunoassay, also manufactured by Abbott Laboratories. Aliquots of a drug-free serum pool were supplemented with aqueous extract of Chan Su or Lu-Shen-Wan and apparent digoxin concentrations were measured using all three digoxin assays. Significant crossreactivity of Chan Su and Lu-Shen-Wan was observed with the new Digoxin III assay. Moreover, when mice were fed with Chan Su or Lu-Shen-Wan, significant apparent digoxin concentrations were also observed in the sera of mice using the Digoxin III assay indicating that such interferences are also present in vivo. When serum pools prepared from patients receiving digoxin were further supplemented with Chan Su or Lu-Shen-Wan extract, falsely elevated digoxin values were observed with both Digoxin III and fluorescence polarization immunoassay, but digoxin values were falsely lowered using the Digoxin II assay. For example, when one aliquot of Digoxin Serum Pool 1 containing 0.94 ng/mL of digoxin was supplemented with 5.0 microg/mL of Chan Su extract, the digoxin concentration was falsely elevated to 6.60 ng/mL as measured by the Digoxin III assay and 6.99 as measured by the fluorescence polarization immunoassay assay. In contrast, the observed digoxin value was falsely lowered to 0.72 ng/mL using the Digoxin II assay. Interference of Chan Su and Lu-Shen-Wan in the Digoxin III assay cannot be eliminated by monitoring free digoxin concentrations. Digibind neutralizes digoxin-like immunoreactive components of Chan Su and such effect can be monitored by measuring apparent free digoxin concentrations using the Digoxin III assay. We conclude the both Chan Su and Lu-Shen-Wan significantly interfere with serum digoxin measurements by the new Digoxin III assay.     

False-positive serum digoxin concentrations determined by three digoxin assays in patients with liver disease

The incidence and magnitude of false-positive serum digoxin concentrations (SDCs) determined by three digoxin assays in patients with liver disease were studied. Patients with biochemical evidence of liver disease were enrolled in the study if they had never received a cardiac glycoside, were not pregnant, were not receiving spironolactone, did not have moderate to severe renal impairment, and did not have transient elevations in liver function test results. Blood specimens from each patient were assayed for apparent SDCs in triplicate using a fluorescence polarization immunoassay (FPIA, TDx Digoxin II, Abbott) and a digoxin radioimmunoassay (RIA, GammaCoat I125, Clinical Assays) and in duplicate using a fluorometric enzyme immunoassay (Dade Stratus, American Dade). Forty-two patients met the study criteria. The percentage of patients exhibiting detectable apparent SDCs (greater than or equal to 0.2 ng/mL) was 57% with RIA, 55% with FPIA, and 28% with the fluorometric enzyme immunoassay. Apparent SDCs ranged from 0.2 to 0.6 ng/mL (RIA), 0.2 to 1.56 ng/mL (FPIA), and 0.2 to 0.38 ng/mL (fluorometric enzyme immunoassay). Values obtained using the fluorometric enzyme immunoassay were significantly different from the apparent SDCs determined using RIA and FPIA; however, no significant difference was found between the values obtained using RIA and FPIA. Significant correlations were found between the apparent SDCs determined using RIA and serum bilirubin values and between the apparent SDCs determined using the fluorometric enzyme immunoassay and alkaline phosphatase values. Of the three assay methods tested, the fluorometric enzyme immunoassay showed the least cross-sensitivity to digoxin-like immunoreactive substance (DLIS).(ABSTRACT TRUNCATED AT 250 WORDS)     

New enzyme-linked chemiluminescent immunosorbent digoxin assay is free from interference of Chinese medicine DanShen

DanShen is a traditional Chinese medicine indicated for cardiovascular diseases. The potential interference of DanShen with serum digoxin measurement was investigated using a new enzyme-linked chemiluminescent immunosorbent (ECLIA) digoxin assay. Aliquots of drug-free serum were supplemented with ethyl acetate extract of DanShen (4 different brands studied), and apparent digoxin concentrations were measured by the ECLIA as well as fluorescence polarization immunoassay (FPIA) and a turbidimetric assay for comparison. Mice were also fed 4 DanShen preparations and apparent digoxin concentrations were subsequently measured. In another experiment, serum pools containing digoxin were further supplemented with DanShen extracts and digoxin concentrations were measured again by all 3 assays. No apparent digoxin concentration was observed when aliquots of drug-free serum pools were supplemented with DanShen and digoxin concentrations were measured by the ECLIA or the turbidimetric assay. In contrast, significant apparent digoxin concentrations were observed using FPIA, and the highest apparent digoxin concentration was observed with brand 4 of DanShen extract. Similarly, when mice were fed with this herb, significant apparent digoxin concentrations were also observed using FPIA, but neither ECLIA nor turbidimetric assay showed any apparent digoxin concentration. When aliquots of digoxin pool were further supplemented with various DanShen extract, the apparent digoxin concentrations were significantly increased when FPIA was used. In contrast, digoxin concentrations in the presence of DanShen extract compared well with the digoxin concentration of the original pool when ECLIA or turbidimetric assay was used. We conclude that DanShen does not interfere with serum digoxin measurement using a more recently released ECLIA digoxin assay.     

Predictive performance study of two digoxin assays in subjects with various degrees of renal function

This prospective study was conducted to compare the predictive performance of fluorescence polarization immunoassay (FPIA, Abbott TDx Digoxin II) and radioimmunoassay (RIA, Kallestad Labs) with combined low-pressure liquid chromatography/RIA (LPLC/RIA) digoxin assay in measuring 15-17 serum digoxin concentrations (SDC) obtained after a single 10 microg/kg intravenous digoxin dose in patients with various degrees of renal function and at different SDC ranges. Eighteen men and women were stratified into 3 age- and gender-matched groups based upon renal function [N = 6 in each, group I (Cl(cr) < 10 mL/min), group II (Cl(cr) = 10-50 mL/min), and group III (Cl(cr) > 50 mL/min)]. Serum digoxin concentrations were measured at time zero; at 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, and 12 hours; and at 2, 3, 4, and 5-7 days after the digoxin dose, using the three different digoxin assays. TDx Digoxin II was unbiased [mean error -0.09 (95% CI -0.19, 0.01)] and RIA biased [mean error -0.29 (95% CI -0.36, -0.21)] to over-predict SDC by 14.2%. In group I patients, the analysis revealed a bias to over-predict SDC by 6.0% for TDx Digoxin II [mean error -0.16 (95% CI -0.29, -0.07)] and an unbiased performance by RIA. In groups II and III, both TDx Digoxin II and RIA showed biased performance, the mean magnitude of bias was low (< 20%). For intermediate SDC range (> 0.5 ng/mL and < or = 3.0 ng/mL), TDx Digoxin II was unbiased in predicting SDC, whereas RIA was biased to under-predict SDC [mean error 0.13 (95% CI 0.10, 0.16)] by 9.9%. The magnitude of bias observed in all cases was less than 20%. Both assays, TDx Digoxin II and RIA, imprecisely measured SDC for all samples combined, different groups and SDC ranges. In all time-paired samples, TDx Digoxin II (FPIA) performed better than the RIA. In conclusion, the magnitude of bias observed with either assay at different groups and SDC ranges was not likely to be clinically relevant. Therefore, either assay may be used to measure SDC in clinical practice.     

Analytic performance evaluation of a new turbidimetric immunoassay for phenytoin on the ADVIA 1650 analyzer: effect of phenytoin metabolite and analogue

Phenytoin is an anticonvulsant that requires therapeutic drug monitoring. Recently, Bayer HealthCare, Diagnostics Division released a turbidimetric immunoassay of phenytoin on the ADVIA 1650 analyzer. We evaluated the analytic performance of this assay by comparing values obtained in 52 patients receiving Phenytoin using this new assay with the values obtained by using a widely used fluorescence polarization immunoassay (FPIA). The new turbidimetric immunoassay for phenytoin showed the following imprecision with the low, medium, and high controls: total CV of 5.2% (mean 4.81 microg/mL), 3.7% (mean 16.24 microg/mL), and 4.1% (mean 22.65 microg/mL), respectively. The detection limit of the assay was 0.79 microg/mL, and the assay was linear up to a phenytoin concentration of 46.1 microg/mL. The assay showed excellent dilution recovery and recovery of spiked samples (mean recovery 101.4% and 94.4%, respectively). We observed an excellent correlation between the values obtained by the FPIA (x-axis) assay and the new turbidimetric (y-axis) assay (y=1.06 x-0.61, r=0.98, n=52). We also determined the cross-reactivity of 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), a major metabolite of phenytoin, and of oxaprozine, an analogue with a similar chemical structure to phenytoin, in both phenytoin assays. Both assays showed almost no cross-reactivity to oxaprozine and only small (5%-8%) cross-reactivity to HPPH. We also found that the turbidimetric assay was free from interference at least up to 1200 mg/dL of hemolysis, 30 mg/dL of free bilirubin, 34.5 mg/dL of conjugated bilirubin, and 750 mg/dL of triglyceride (Intralipid). When a drug-free serum was followed by a serum sample containing 38.5 microg/mL of phenytoin, no sample probe carryover effect was observed. We conclude that the new turbidimetric assay can be used for routine monitoring of phenytoin in clinical laboratories.     

老年心功能不全患者血清地高辛样免疫活性物质测定

采用荧光极化免疫分析法,检测了15例老年心功能不全病人血清地高辛样免疫活性物质(DLIS)浓度。以TDx仪最低检测限0.256nmol·L^-1为阳性检测标准,DLIS检出阳性率为46.7%(7/15),浓度范围0.26～1.52nmol·L^-1,平均浓度0.55±0.44nmol·L^-1;其中1例病人血清DLIS浓度高达1.52nmol·L^-1。结果表明老年心功能不全患者血清中含有较高水平的DLIS;提示对这些患者若因病情需要而用地高辛治疗时,其后的血浓度测定结果易引起“过高估计”,应当谨慎地评价地高辛血浓度测定结果。     

Monitoring urinary excretion of cannabinoids by fluorescence-polarization immunoassay: a cannabinoid-to-creatinine ratio study

Drug testing in substance abuse treatment programs is focused on urine analysis of parent drugs and major metabolites. Huestis reported that serial monitoring of the major urinary cannabinoid metabolite (delta9-THC-COOH)-to-creatinine ratios in paired urine specimens (collected at least 24 hours apart) could differentiate new marijuana or hashish use from residual cannabinoid metabolite excretion in urine after previous drug use. Subjects with a history of chronic marijuana use were screened for cannabinoids in urine over several months by an enzyme immunoassay (EMIT) with a cut-off value of 50 ng/mL. Presumptive positive specimens were confirmed by gas chromatography-mass spectrometry (GC-MS) for delta9-THC-COOH with a cut-off value of 15 ng/mL. The objective of this study was to determine whether a semiquantitative cannabinoids immunoassay (corrected for creatinine concentration) could differentiate new marijuana use from residual cannabinoid excretion in chronic users of marijuana or hashish compared with GC-MS. The criterion for new marijuana use was a cannabinoid-to-creatinine ratio > or =0.5 (dividing the immunoassay quantitative result to creatinine ratio of specimen 2 by the specimen 1 ratio, specimen 3 by the specimen 2 ratio, etc.). Urine specimens were analyzed by fluorescence-polarization immunoassay (FPIA) on an Abbott TDxFLx analyzer after analysis by GC-MS. In 90 urine specimens (group A) with delta9-THC-COOH values determined by GC-MS, the mean delta9-THC-COOH concentration was 44.4 ng/mL (range, 16-100), and the mean FPIA total cannabinoids value was 91.7 ng/mL (range, 21-204 ng/mL) with a correlation coefficient of 0.993 (group A). In 111 specimens (group B), the mean delta9-THC-COOH concentration was 361 ng/mL (range, 101-960 ng/mL). The mean FPIA value was 657 ng/mL (range, 211-1,270 ng/mL), and the correlation coefficient of the B series was 0.975. Percent cross-reactivity for delta9-THC-COOH standards prepared in drug-free urine by FPIA was 82% at 25 ng/mL, 45% at 50 ng/mL, and 50% at 100 ng/mL. Overall, there was 89% agreement (132 of 148 specimens) between FPIA and GC-MS. In 16 of 148 specimens, however, the FPIA and GC-MS paired urine data did not agree. The sensitivity of the FPIA assay was 95.3%, and the specificity was 44.4%. The authors conclude that FPIA cannabinoid analysis should be further evaluated as an alternative to GC-MS quantitation to help distinguish new marijuana use from residual marijuana metabolite excretion in clinical drug treatment programs.     

Effect of digoxin fab antibody on the measurement of total and free digitoxin by fluorescence polarization and a new chemiluminescent immunoassay

Digoxin fab antibody (Digibind; Burroughs Wellcome, Research Triangle Park, NC, USA) is used in the treatment of digoxin overdose. The effect of digibind on the measurement of total and free digoxin has been extensively studied. However, the effect of digibind on digitoxin measurements has not been studied thoroughly. The authors studied the effect of digibind on the measurement of total and free digitoxin in vitro using the fluorescence polarization immunoassay and a new chemiluminescent immunoassay. We also studied the capability of digibind to bind digitoxigenin, the major aglycon metabolite of digitoxin. Digibind neutralized both digitoxin and digitoxigenin in vitro, as evidenced by significant reductions in free digitoxin and digitoxigenin (measured as digitoxin equivalent) concentrations. Digibind caused negative interference in the measurement of total digitoxin concentrations by both fluorescence polarization and chemiluminescent assays. However, the magnitude of negative interference was significantly higher with the chemiluminescent assay. For example, in a serum pool supplemented with 80 ng/mL of digitoxin, the concentrations of total and free digitoxin measured by the fluorescence polarization immunoassay were 82.1 ng/mL and 3.3 ng/mL respectively. In the presence of 5 microg/mL of Digibind, the corresponding total and free digitoxin concentrations were 73.9 ng/mL and none detected, respectively. In another serum pool supplemented with 70 ng/mL of digitoxin, the concentrations of total and free digitoxin as measured by the chemiluminescent assay were 69.1 ng/mL and 3.8 ng/mL, respectively. In the presence of 5 microg/mL of Digibind, the corresponding total and free digitoxin concentrations were 29.0 ng/mL and none detected, respectively. Because this effect may also occur in vivo, the progress of Digibind therapy in treating a patient with digitoxin overdose may be monitored by measuring the free digitoxin concentrations.     

Effect of spironolactone, potassium canrenoate and their common metabolite canrenone on serum digoxin measurement by digoxin III, a new digoxin immunoassay

Spironolactone and potassium canrenoate (aldosterone antagonist diuretics) are often used with digoxin in clinical practice. It has been well documented in the literature that spironolactone, potassium canrenoate, and their common metabolite canrenone cross-react with several digoxin immunoassays at concentrations expected after therapeutic usage of these drugs and falsely elevate or lower serum digoxin concentrations. Recently, Abbott Laboratories marketed a new Digoxin III immunoassay for application on the AxSYM analyzer. We studied the potential interference of these compounds with this new digoxin assay. The Tina-quant assay was used as the reference method because spironolactone, potassium canrenoate, and canrenone do not interfere with serum digoxin measurement using this assay. Aliquots of drug-free serum were supplemented with therapeutic and above therapeutic concentrations of spironolactone, canrenone, and potassium canrenoate, and apparent digoxin concentrations were measured using the Digoxin III assay and Tina-quant assay. Significant apparent digoxin concentrations were observed when the Digoxin III digoxin assay was used, but no apparent digoxin levels was observed using the Tina-quant assay. When serum pools prepared from patients receiving digoxin were further supplemented with these compounds in concentrations expected in sera of patients receiving these medications, falsely elevated digoxin levels were observed using Digoxin III assay, but no statistically significant change was observed using the Tina-quant assay. We conclude that spironolactone, potassium canrenoate, and their common metabolite canrenone interfere with the serum digoxin measurements using the new Digoxin III assay.     

Comparison of digoxin analysis by high-performance liquid chromatography/post-column derivatization and fluorescence polarization immunoassay

1. This study compared the analysis of digoxin using a high-performance liquid chromatographic post-column derivatization (HPLC-PC) assay and the TDx fluorescence polarization immunoassay (FPIA). 2. Serum obtained from 15 digitalized patients showed higher mean digoxin levels with the FPIA method as compared to the HPLC-PC procedure such that the mean HPLC-PC/FPIA ratio was 0.91 +/- 0.14 (mean +/- SD). Demonstrated cross-reactivity of digoxin metabolites with the FPIA is probably responsible for this observation. 3. Cross-reactivity of the immunoassay towards endogenous material present in serum samples from certain patient groups was an even greater problem, with apparent 'digoxin' serum concentrations in untreated hepatic failure patients being within the therapeutic range for digoxin. 4. The HPLC-PC method did not suffer from such interference and would therefore provide more accurate values for patients where high levels of interference could contribute to false digoxin levels.     

Effect of digoxin Fab antibodies on five digoxin immunoassays

Following digoxin Fab antibody (FAB) administration in digitalis-toxic patients, total serum digoxin concentrations (SDCS) become elevated, but do not correlate with pharmacologic activity. In an attempt to accurately measure free (pharmacologically active) SDC in the presence of FAB, we assessed the utility of five digoxin immunoassays: fluorescence polarization immunoassay (FPIA), ultrafiltration with FPIA (ULTRA-FPIA), enzyme multiplied immunoassay (EMIT), radioimmunoassay (RIA), and American Dade's STRATUS (STRATUS). To normal human serum samples containing 2 and 4 ng/ml of digoxin, FAB was added in escalating quantities of 0-1.9 micrograms. In addition, 1.9 micrograms of FAB was added to two serum samples containing no digoxin. SDCS reported by FPIA for each 2 ng/ml samples, in order of ascending FAB doses, were 2.08, 1.94, 2.02, 1.99, 1.95, and 1.93 ng/ml, while the SDCS from the ULTRA-FPIA were 2.00, 1.76, 1.56, 1.36, 1.16, and 0.98 ng/ml. Results similar to the ULTRA-FPIA were obtained with the STRATUS, RIA, and EMIT, although the SDCS from the EMIT (p less than 0.05) samples exhibited greater fluctuation. The 4 ng/ml samples demonstrated similar patterns among the assays although no statistical differences were noticed between EMIT and ULTRA-FPIA. Samples containing FAB without digoxin only adversely affected the RIA, which reported mean SDCS from the two identically prepared samples of 5.8 and 7.8 ng/ml. Except for the FPIA, the SDC measured by the assays directly correlated with the amount of FAB in the sample, demonstrating the ability of these assays to measure free SDC.     

Digitalis-like immunoreactive substances in maternal and umbilical cord plasma: a comparative sensitivity study of fluorescence polarization immunoassay and microparticle enzyme immunoassay

Digitalis-like immunoreactive substances (DLIS) obtained from maternal and umbilical cord plasma at delivery were measured by fluorescence polarization immunoassay (FPIA; TDX, Abbott) and microparticle enzyme immunoassay (MEIA; IMX, Abbott). In each sample, concentrations of dehydroepiandrosterone, dehydroepiandrosterone sulfate, estradiol, estriol, hydrocortisone, progesterone, and testosterone were measured by radioimmunoassay, and cross-reaction tests of DLIS with these substances were conducted. By FPIA, the concentration of DLIS in umbilical cord plasma (0.55 +/- 0.22 ng/mL) was significantly higher than that in maternal plasma (0.23 +/- 0.11 ng/mL). In the cross-reaction tests, when the concentration of dehydroepiandrosterone sulfate was higher than 1.0 microg/mL or that of progesterone was higher than 0.5 microg/mL, DLIS were detected by FPIA. However, DLIS were not found either in the samples or in the cross-reaction tests by MEIA. By radioimmunoassay, there was no significant difference in the dehydroepiandrosterone sulfate concentration between the maternal plasma (2,917 +/- 1,001 ng/mL) and the umbilical cord plasma (1,957 +/- 376 ng/mL). The progesterone concentration in the umbilical cord plasma (310.0 +/- 85.7 ng/mL) was significantly higher than that in the maternal plasma (126.4 +/- 38.5 ng/mL). These results suggest that dehydroepiandrosterone sulfate in maternal plasma and progesterone in maternal and umbilical cord plasma may be measured as digoxin by FPIA.     

Determination of salivary digoxin with a dry strip immunometric assay.

Analysis of salivary digoxin using a rapid dry chemistry, enzyme-labeled immunometric assay (ELIA) was compared with fluorescence polarization immunoassay (FPIA). Saliva and serum samples were obtained from 40 hospitalized patients who were taking digoxin chronically and from 8 patients just prior to treatment with digoxin. Unstimulated saliva samples were collected from 20 patients; however, saliva volumes from 10 pediatric patients were inadequate to permit analysis by FPIA, and 1 other had unmeasurable concentrations by both methods. Stimulated saliva was collected by having patients chew a small piece of Parafilm for 1-2 min. Salivary digoxin was analyzed using the same procedure recommended for serum digoxin by each manufacturer. There were no significant differences found between ELIA and FPIA determinations of unstimulated or stimulated salivary digoxin, serum digoxin, or saliva/serum concentration ratios. The saliva/serum ratio of the unstimulated group was approximately twice that of the stimulated group (p less than 0.01) by both methods, suggesting that salivary digoxin concentration decreases with increased saliva production rate. Excellent correlations were found between ELIA and FPIA salivary digoxin concentrations and between stimulated saliva and serum concentrations by both assays. Weaker correlations were observed between unstimulated saliva and serum concentrations. There was no evidence of assay interference with either method in eight nondigitalized patients, each taking an average of 6.5 medications. The ELIA appears to provide equivalent results compared with the FPIA for the determination of salivary digoxin concentration. Further investigations are needed before salivary digoxin concentration monitoring can be recommended as an acceptable alternative to serum monitoring.