Effect of Brazilian, Indian, Siberian, Asian, and North American ginseng on serum digoxin measurement by immunoassays and binding of digoxin-like immunoreactive components of ginseng with Fab fragment of antidigoxin antibody (Digibind)

We compared Brazilian, Indian, Siberian, Asian, and North American ginseng for potential interference with 3 digoxin immunoassays: fluorescence polarization (FPIA), microparticle enzyme (MEIA), and Tina-quant (Roche Diagnostics, Indianapolis, IN). We supplemented aliquots of a drug-free serum pool with ginseng extracts representing expected in vivo concentrations and overdose. We observed apparent digoxin-like immunoreactivity with FPIA, modest immunoreactivity with MEIA, and no apparent digoxin immunoreactivity with the Tina-quant with all ginsengs except Brazilian, which showed no immunoreactivity with any assay. When aliquots of serum pools prepared from patients receiving digoxin were supplemented with ginsengs, we observed falsely elevated digoxin values with FPIA, falsely lower digoxin values (negative interference) with MEIA, and no interference with the Tina-quant. Digoxin-like immunoreactive components of various ginsengs have moderate protein binding; monitoring free digoxin concentrations does not eliminate such interference. We also observed that Digibind (Burroughs Wellcome, Research Triangle Park, NC) can bind free digoxin-like immunoreactive components of ginsengs; such effects can be monitored by measuring apparent free digoxin concentrations. Indian, Asian, and North American ginsengs interfere with serum digoxin measurement by FPIA and MEIA; the Tina-quant is free of such interference. Digibind can bind free digoxin-like immunoreactive components of ginseng.     

Positive and negative interference of the Chinese medicine Chan Su in serum digoxin measurement. Elimination of interference by using a monoclonal chemiluminescent digoxin assay or monitoring free digoxin concentration

An over-the-counter Chinese medicine, Chan Su, is used as a cardiotonic agent. We demonstrated significant digoxin-like immunoreactivity in various organic and aqueous extracts of Chan Su. For example, when a 20-microL aliquot of an aqueous extract of Chan Su powder (1 mg/mL) was added to a 2-mL aliquot of a drug-free serum, the observed digoxin-like immunoreactivity was 2.76 ng/mL (3.53 nmol/L) digoxin equivalent using the fluorescence polarization immunoassay (FPIA). The magnitude of interference was much lower (0.94 ng/mL [1.20 nmol/L]) with the microparticle enzyme immunoassay (MEIA), and no interference was observed with the chemiluminescent assay (CLIA). We also observed a significant positive interference of the extract with the serum digoxin measurement using FPIA. In contrast, we observed a negative interference (falsely lowered digoxin concentration) of the extract in the serum digoxin measurement with the MEIA. The extract had no effect on the serum digoxin measurement with the CLIA. By taking advantage of the high protein binding of Chan Su and only 25% protein binding of digoxin, we further demonstrated that positive interference of Chan Su in the FPIA and negative interference of Chan Su in the MEIA of digoxin could be eliminated by monitoring the free digoxin concentration.     

Neutralization of free digoxin-like immunoreactive components of oriental medicines Dan Shen and Lu-Shen-Wan by the Fab fragment of antidigoxin antibody (Digibind)

Dan Shen and Lu-Shen-Wan, traditional Chinese medicines used as remedies for heart diseases, demonstrate digoxin-like immunoreactivity. The digoxin-like immunoreactive components of Lu-Shen-Wan show approximately 55% protein binding, while Dan Shen demonstrates concentration-dependent protein binding (68% bound at lower concentrations but only 25% bound at higher concentrations). Because Dan Shen and Lu-Shen-Wan can cause substantial toxic effects in patients, we studied the potential use of Digibind (Fab fragment of polyclonal antidigoxin antibody; Burroughs Wellcome, Research Triangle Park, NC) for neutralizing the pharmacologically active free fractions of Dan Shen and Lu-Shen-Wan. Drug-free serum pools were supplemented with Dan Shen or Lu-Shen-Wan to achieve apparent digoxin concentrations expected in severe overdoses. Aliquots of supplemented serum pools were supplemented further with aqueous Digibind solution to achieve final Digibind concentrations between 5 and 20 microg/mL (expected in vivo range in patients overdosed with digoxin and being treated with Digibind). We observed complete removal of the free apparent digoxin in the presence of Digibind for Dan Shen and Lu-Shen-Wan. Digibind binds free digoxin-like immunoreactive components of Dan Shen and Lu-Shen-Wan in vitro.     

Effect of Asian and Siberian ginseng on serum digoxin measurement by five digoxin immunoassays. Significant variation in digoxin-like immunoreactivity among commercial ginsengs

Asian and Siberian ginsengs contain glycosides with structural similarities to digoxin. We studied potential interference of ginseng in 5 digoxin immunoassays in 3 Asian (2 liquid extracts, 1 capsule) and 3 Siberian ginseng preparations (1 liquid extract, 2 capsules). With the fluorescence polarization immunoassay (FPIA), we observed apparent digoxin activity in 1 Asian liquid preparation and in the liquid extract and 1 capsule form of Siberian ginseng. In mice fed ginseng, we observed digoxin activities in the serum (Asian, 0.48-0.68 ng/mL [0.6-0.9 nmol/L]; Siberian, 0.20-0.47 ng/mL [0.3-0.6 nmol/L]), indicating that such interferences also occur in vivo. Serum pools prepared from samples from patients receiving digoxin and then supplemented with Asian or Siberian ginseng showed falsely increased digoxin values using the FPIA (e.g., for Asian ginseng, 1.54 ng/mL [2.0 nmol/L] vs control value, 1.10 ng/mL [1.4 nmol/L]) and falsely decreased values using the microparticle enzyme immunoassay (MEIA; 0.73 ng/mL [0.9 nmol/L] vs control value, 1.04 ng/mL [1.3 nmol/L]). Digoxin-like immunoreactive substances (DLISs) showed synergistic effects with ginsengs in interfering with the FPIA and MEIA for digoxin. No interference was observed with 3 other digoxin assays, even in the presence of elevated DLISs.     

Suppression of total digoxin concentrations by digoxin-like immunoreactive substances in the MEIA digoxin assay. Elimination of negative interference by monitoring free digoxin concentrations

Digoxin-like immunoreactive substances (DLIS) cross-react with antidigoxin antibody and falsely elevate immunoassay-measured total digoxin concentrations. The fluorescence polarization immunoassay (FPIA) for digoxin showed high cross-reactivity with DLIS, but a new microparticle enzyme immunoassay (MEIA) had low cross-reactivity. The concentration of digoxin in the presence of DLIS was falsely lowered (negative interference) when measured by MEIA. We prepared the following serum pools: 2 normal (no DLIS), 2 from patients with uremia, and 3 from patients with liver disease (high DLIS). No patients received digoxin or digitoxin. When normal pools were supplemented with known concentrations of digoxin, total and free concentrations measured by both assays were comparable, but when liver and uremic pools containing high DLIS were supplemented with digoxin, the measured total digoxin concentrations were lower by MEIA and higher by FPIA. However, by taking advantage of 25% protein binding of digoxin and high protein binding of DLIS, free digoxin levels were not affected by DLIS. In 2 patients receiving digoxin but without volume expansion, total and free digoxin concentrations measured by both assays were comparable; in the 2 volume-expanded patients, only free digoxin concentrations were comparable. Monitoring free digoxin concentration can eliminate negative interference of DLIS in the MEIA for digoxin.     

Interference of carbamazepine and carbamazepine 10,11-epoxide in the fluorescence polarization immunoassay for tricyclic antidepressants: estimation of the true tricyclic antidepressant concentration in the presence of carbamazepine using a mathematical model

We evaluated effects of carbamazepine and its metabolite, carbamazepine 10,11-epoxide, on the measurement of tricyclic antidepressant (TCA) concentrations in serum using the fluorescence polarization immunoassay (FPIA). We determined apparent TCA concentrations in 30 patients who were receiving carbamazepine but no TCAs. Carbamazepine concentrations ranged from 1.4 to 20.9 microg/mL (5.9-88.4 micromol/L); the observed apparent TCA concentrations ranged from 31.8 to 130.1 ng/mL (113.4-463.9 micromol/L). When aliquots of the drug-free serum pool were supplemented with known concentrations of carbamazepine or its metabolite, we observed significant apparent TCA concentrations using the FPIA; however, interference of carbamazepine was more than 3-fold more than its metabolite. When serum pools prepared from patients receiving TCA but no anticonvulsant medications were supplemented with known amounts of carbamazepine or its metabolite, we observed falsely elevated TCA concentrations. We formulated an equation to calculate the apparent TCA concentration from known carbamazepine concentrations. If carbamazepine and TCAs are present in a specimen, the true TCA concentration can be estimated by subtracting the calculated TCA concentration (due to carbamazepine) from the observed TCA concentration as measured by the TCA FPIA. This mathematical modeling is feasible because TCAs, even at very high concentrations, showed no interference with the carbamazepine FPIA.     

A physician's office-based digoxin assay (Seralyzer) evaluated for interference by endogenous digoxin-like immunoreactive factors

A digoxin test for a physician's office based-chemistry analyzer (Ames Seralyzer) was evaluated for possible interference by digoxin-like immunoreactive factors (DLIF). Sera from patients likely to have high concentrations of DLIF (renal and hepatic patients, pregnant women, and neonates) as well as from normal patients and umbilical cord blood were analysed by the Seralyzer digoxin immunoassay and by a fluorescence polarization digoxin immunoassay (Abbott TDx) known to detect DLIF. For all patients who were not taking digoxin (n = 85) only four patients (4.7 percent) measured apparent digoxin values greater than 0.2 ng per mL by the Seralyzer compared to 64 (75 percent) by the TDx analyzer. Measurements of DLIF from adrenal extracts demonstrated a 17-fold greater potency for detection of DLIF by the TDx (2.9 ng per mL) compared to the Seralyzer technique (0.18 ng per mL). However, recovery data suggest that the presence of digoxin reduces the potency of DLIF interference as a function of increasing digoxin concentrations especially for the TDx assay. This diminished DLIF crossreactivity in the presence of digoxin is one explanation for the comparable correlation observed for both non-renal and renal failure patients taking digoxin when measured by these two immunoassays.     

Effects of digoxin immune Fab (ovine) on digoxin immunoassays

Digoxin immune Fab (ovine) (Digibind) is a preparation of Fab fragments of antidigoxin antibodies that is used as an antidote for severe digitalis toxicity. To assess the effects of its presence on digoxin immunoassays, specimens were prepared that simulated typical samples from treated patients and were distributed to a number of laboratories for analysis as unknowns. The antidote interfered to a varying extent with all assays reported. The results suggest that most competitive immunoassays will yield results that bear little relation to concentrations of total digoxin or free digoxin (i.e., digoxin not bound to the antidote). The aca, Dimension, and CEDIA sequential immunoassays moderately overestimated the free digoxin concentration. Free digoxin levels appeared to be only slightly overestimated by the Aria, Stratus, and Syva assays. The TDx Digoxin II assay yielded slight to moderate underestimates of the total digoxin concentration.     

Antidigoxin Fab fragments and digoxin monitoring: a challenge for the biologist

Following administration of anti-digoxin Fab fragments, monitoring unbound digoxin concentrations may help ensure appropriate dosing, and prevent recrudescent toxicity. Ultrafiltration by using Centrifree system and measurement of digoxin in the ultrafiltrate is considered as reference technique. However, ultrafiltration method is cumbersome, costly, and some immunoassays are affected by matrix differences. Another approach is to analyse the serum directly by digoxin immunoassays without ultrafiltering it. The validity of results obtained depends on the architecture of the immunoassay and the amount of Fab in the sample. The old radioimmunoassays and usually the other competitive immunoassays give inaccurate results. The fluorescence polarization immunoassay (FPIA) slightly underestimates the total digoxin concentrations. Total digoxin levels obtained at 24 hours and 48 hours after treatment permit measurement of the half-life of digoxin Fab complexes and can be used to estimate when the patient can be redigitalized, if necessary. The sequential immunoassays usually overestimate the free digoxin concentrations. The differences observed are >25% and cannot be explained solely by albumin binding (normal range, 20% +/- 5%). To date, ultrafiltration remains the best strategy for accurate determination of digoxin concentrations in the presence of antidigoxin Fab fragments.     

Digoxin assay: results of 10 years of intralaboratory controls

Serum digoxin measurement is often performed in medical laboratories. A professional association specialized in quality control, based in Lyon, has been organizing punctual controls of medication measurement for the past ten years. The results are analysed in term of intra and inter-technique precision, difference between methods and specificity in regard to endogenous or exogenous interfering substances. Methods have changed with a quasi disappearance of the methods used ten years ago (FPIA, EMIT) and introduction of new technologies on recent immunoanalysis automates. The results observed with the different instruments are similar. Reproductibility has not changed over ten years. Some difficulties remain in the measurement of low concentrations of digoxin. Many substances interfere in digoxin measurement : digoxigenine (inactive metabolite), endogenous digoxin-like immunoreactive factors, spironolacton, antidigoxin antibodies used for treatment of digitalic intoxications. These interferences depend on the method which is used, but it is essential to know them in order to interpret the results correctly.     

Digoxin and digoxin-like immunoreactive factors (DLIF) modulate the release of pro-inflammatory cytokines

Objective:   Cardiac glycosides such as digoxin and their endogenous counterpart digoxin-like immunoreactive factor (DLIF) may possess anti-inflammatory properties. Methods:  Pro-inflammatory cytokines from human peripheral blood mononuclear cells (PBMC) were measured by ELISA using specific antibodies. Immunocytochemistry was used to localize NF-kB. Results:   Non-stimulated PBMC constitutively secreted minimum amounts of cytokines. LPS (1 mg/L) stimulation lead to steep increases in TNF-alpha, IL-6 and IL-8 concentrations with peak rises at 8 h. An 8 h delay was observed for IL-10. Increases in IL-10 were sustained for18 h period. Significant inhibition (P > 0.05) of TNF-alpha, IL-6 and IL-8 at non-toxic of digoxin concentration (< 100 nM) and DLIF (10 nM digoxin equivalent (de)) was observed whereas no such effect was seen for IL-10. Inhibition of the degradation of activated NF-kB in the PBMC was observed with the indicated concentrations of digoxin, DLIF or Pyrrolidine dithiocarbamate (PDTC). Conclusion:   Digoxin and DLIF inhibit the release of proinflammatory cytokines from PBMC via NF-kB-dependent pathway suggesting an anti-inflammatory effect. Received 30 December 2007; returned for revision 22 April 2008; received from final revision 24 April 2008; accepted by G. Wallace 22 May 2008 The first two authors contributed equally to this work.     

Comparison of non-digitalis binding properties of digoxin-specific Fabs using direct binding methods

Digibind and DigiFab are commercial formulations of polyclonal, ovine, digoxin-specific Fabs in clinical use for treatment of digoxin intoxication. Of interest for extending its use to other clinical indications, Digibind has also been reported to neutralize the effect of endogenous digoxin-like molecules, including ouabain, that are linked to clinical disorders ranging from preeclampsia to congestive heart failure. Although Digibind and DigiFab are equivalent in their digoxin-binding activity, the antigens used to produce these Fabs are different. We therefore explored, using native (3)H-digoxin and (3)H-ouabain in four different types of solution-phase binding methods, whether they might exhibit different profiles with respect to ouabain and other digoxin-like factors. Consistent with previous results, both Fab preparations bound digoxin with the same affinities and capacities. However, (3)H-ouabain was found to bind with high affinity only to Fab sub-populations present in both products. Interestingly, this sub-population was twice as large for Digibind compared to DigiFab. Competition experiments also showed differences in specificity within Fab sub-populations. Therefore, the equivalence in digoxin-binding activity of the two Fab preparations does not extend to ouabain-binding capacity and Fab specificity, with implications for clinical differentiation between the preparations in treatment of disorders related to control of non-digoxin cardenolides. The existence of a small but perhaps clinically relevant sub-population of antibodies was detected using specific radioligands. This sub-population could not have been detected nor quantified using standard cross-reactivity in an ELISA assay.     

Digoxin-like and digitoxin-like immunoreactive substances in elderly people. Impact on therapeutic drug monitoring of digoxin and digitoxin concentrations

We compared digoxin-like (DLIS) and digitoxin-like (DTLIS) immunoreactive substance concentrations for 30 people older than 65 years with those for 25 people younger than 50. None received digoxin or had liver disease, uremia, or volume expansion. We found no DTLIS in any specimen, and only 1 specimen from an elderly person demonstrated a low DLIS concentration. In addition, for 22 non-volume expanded patients (8 younger than 50 years and 14 older than 65) receiving digoxin, the fluorescence polarization (FPIA) and the microparticle enzyme (MEIA) immunoassays revealed comparable serum digoxin concentrations, indicating an insufficient DLIS concentration to interfere with digoxin immunoassay results. Therefore, elderly people who are not volume expanded do not have elevated DLIS or DTLIS concentrations. Furthermore, for patients with liver disease or uremia (18 older than 65 years and 20 younger than 50), the DLIS and DTLIS concentrations were elevated. Finally, for 5 patients with liver disease who received digoxin, serum digoxin concentrations were lower by MEIA and higher by FPIA, indicating the patients had elevated DLIS levels that interfered with the assays. Elevated DLIS and DTLIS concentrations are associated with volume expansion and not age.     

Assay of digoxin on dry-reagent strips

An automated immunoassay for Digoxin in serum using dry-reagent strips (Stratus Dade) was evaluated, and compared with two others immunoassays (TDX Abbott and ACA Du Pont). Precision, sensitivity and specificity were satisfying, as well as correlation study, but discrepancies observed for some sera proved that these techniques still pose serious problems of specificity or of standardisation.     

Effect of therapeutic digoxin antibodies on digoxin assays

The introduction of therapeutic digoxin antibodies in the treatment of digoxin toxicity has resulted in spurious results in the many assays of cardioglycoside in serum of patients receiving this therapy. An evaluation of currently used assay methods was incorporated into a pilot Ligand Assay Survey Program of the College of American Pathologists to define which assays were unaltered by the presence of a therapeutic digoxin antibody (Digibind). Disparity of affinity constants of the therapeutic and kit antibodies is the most likely explanation for erroneous values. Specific methods are defined that appear to be free of this phenomenon.     

Endogenous and exogenous digoxin-like immunoreactive substances: impact on therapeutic drug monitoring of digoxin

Endogenous digoxin-like immunoreactive substance (DLIS) was first reported in volume-expanded dogs. Its presence has been confirmed in blood, urine, and other body fluids. Elevated DLIS concentrations are encountered in patients with volume-expanded conditions such as uremia, essential hypertension, liver disease, and preeclampsia. DLISs cross-react with antidigoxin antibodies and falsely elevate serum digoxin concentrations, interfering in interpretation of results for therapeutic digoxin monitoring. Falsely lower digoxin values due to the presence of DLISs have been reported. The association of DLISs with volume expansion led to speculation that they could be natriuretic hormones. Several structures have been proposed for DLISs, including nonesterified fatty acid, phospholipid, lysophospholipid, bile acid, bile salt, and steroid. Exogenous DLISs can be found in serum after ingestion of various Chinese medicines and therapy with spironolactone, canrenone, or potassium canrenoate. Like endogenous DLISs, exogenous DLISs interfere with serum digoxin assays, complicating therapeutic digoxin monitoring. However, most reported endogenous and exogenous DLISs are strongly protein-bound while digoxin is weakly protein-bound. Therefore, interference of both endogenous and exogenous DLISs in serum digoxin measurement can be eliminated by monitoring digoxin concentrations in the protein-free ultrafiltrates.     

血浆地高辛浓度危急值的应用与评估

目的 对血浆地高辛浓度危急值进行回顾性分析,为持续改进该项目危急值报告制度提供依据.方法 从实验室信息系统中筛选出血浆地高辛浓度危急值数据,经数据处理,统计出该项目危急值的发生率、天内分布、周内分布、科室分布,评估危急值范围和报告的周转时间.结果 血浆地高辛浓度危急值的发生率占该项目测试数的2.98％,科室分布以心内科为主.83.3％危急值报告的实验室周转时间在3小时之内.评估该项目频数分布图、临床症状符合率后,认为可以继续使用原定的血浆地高辛浓度危急值.结论 定期分析评估（一年一次）,持续改进血浆地高辛浓度危急值报告工作程序,可以提高检验科和临床科室的工作效率和质量,满足患者的安全要求.     

Performance comparison of three assay methods used in fasting and postmethionine load plasma homocysteine determinations from patients with vascular disease

Several methods have been developed for measurement of plasma total homocysteine (tHcy), a proven graded risk factor for cardiovascular diseases. The aim of the study was to compare 3 commonly used clinical methods (enzyme immunoassay, Axis Biochemicals, Oslo, Norway; and fluorescence polarization immunoassay [FPIA], Axis, on the Abbott IMx System, Abbott Laboratories, Abbott Park, IL; and BioRad high-performance liquid chromatography [HPLC], Laboratories, Munich, Germany) for tHcy determination in samples from cardiovascular patients also undergoing methionine load measurement. Only HPLC reached the required coefficient of variation (<6%) for desirable performance for tHcy concentrations ranging between 0.68 and 5.41 mg/L (5 and 40 micromol/L). A higher variability was observed for postmethionine load vs fasting samples, which reached statistical significance for FPIA (P = .0013). The closest agreement between methods was observed between HPLC and FPIA for fasting tHcy concentrations (-0.23 mg/L [-1.69 micromol/L], -30% to 8%).Both immunoassays could be suitable alternatives for laboratories with high workloads when HPLC is not available. Differences among tHcy values must be taken into account when these methods will be used interchangeably for diagnosis and monitoring of treatment.     

地高辛的生物素—链霉亲和素免疫法测定方法的建立

目的：将生物素链霉亲和素免疫反应系统引入地高辛血药浓度监测中，准确地监测地高辛血药浓度。方法：将生物素与抗ＤＩＧＩｇＧ化学连接，ＤＩＧ通过ＨＳＡ与ＨＲＰ连接，建立了生物素链霉亲和素免疫反应系统测定地高辛的竞争反应模型。结果：以０５、１、２、３、５μｇ／Ｌ五个标准点建立了竞争反应曲线，同时将ＢＮＨＳＩｇＧ作了１：４００、１：８００、１：１６００、１：３２００的稀释，探讨了竞争反应曲线的规律性变化，运用流行的四参数曲线拟合法对竞争曲线作了拟合。同时做了方法学评价。结论：流行的生物素链霉亲和素技术可以引入被动的酶联免疫吸附法中，提高反应的灵敏度