Fluorescence polarization immunoassay: can it result in an overestimation of vancomycin in patients not suffering from renal failure?

It has been reported in scientific data that fluorescence polarization immunoassay (FPIA) results in overestimation of vancomycin in patients with renal failure. This overestimation is caused by interference of the degradation product, CDP-1, in this assay. Increases in vancomycin levels have also been reported in patients not suffering from renal failure (nonrenal failure patients) who are receiving vancomycin therapy for approximately 10 days or more. The authors tested whether this increase in vancomycin in nonrenal failure patients is a result of CDP-1 interfering with FPIA or a change in the pharmacokinetics of the drug. Serum vancomycin peak and trough samples were obtained from 10 adult (mean age +/- SD: 55.9 years +/- 17.5) nonrenal failure patients (mean ClCr +/- SD: 76.2 mL/min +/- 29.20) receiving vancomycin therapy for at least 10 days. These peaks and troughs were obtained at steady state and again at approximately 10 days of therapy. All serum samples were analyzed initially by fluorescence polarization immunoassay (FPIA, TDx) (Abbot Diagnostics; Irving, TX) and again by enzyme multiplied immunoassay (EMIT Vancomycin Assay) (Dade Behring; San Jose, CA). Statistical analysis (Wilcoxon signed-rank test) determined that there was no difference between the values obtained from the two assays. This demonstrates that the increase in vancomycin levels is not caused by the accumulation of CDP-1 and may be the result of a change in the pharmacokinetics of the drug.     

Determination of N-desmethylmethsuximide serum concentrations using enzyme-multiplied and fluorescence polarization immunoassays

N-Desmethylmethsuximide (NDM), the active metabolite of the antiepileptic agent methsuximide, has been analyzed by gas-liquid chromatography and high-performance liquid chromatography (HPLC) in the past. This study compares methods using two commercially available immunoassays for ethosuximide, the enzyme multiplied immunoassay technique (EMIT) and fluorescence polarization immunoassay (FPIA), with an HPLC method for the measurement of NDM concentrations in serum. Within-day precision studies, utilizing low therapeutic (15.0 mg/L) and toxic (45.0 mg/L) NDM concentrations (n = 20), resulted in coefficients of variation (CVs) of 4.6 and 4.2%, respectively, for EMIT and 5.4 and 3.2%, respectively, for FPIA. Day-to-day precision studies (n = 10) resulted in CVs of 7.6 and 5.5%, respectively, for EMIT and 3.5 and 2.4%, respectively, for FPIA. No interference was observed from toxic concentrations of acetaminophen, caffeine, carbamazepine, methsuximide, phenobarbital, phensuximide, phenytoin, primidone, salicylate, and valproic acid in the EMIT and FPIA procedures. There was good linear correlation between EMIT and HPLC NDM determinations of 50 patient samples (r = 0.970; y = 0.96 x + 0.03), and a similar correlation between FPIA and HPLC NDM determinations in 48 patient samples (r = 0.975; y = 0.91 x + 1.24). Using ethosuximide reagents, both EMIT and FPIA systems can be adapted to reliably measure NDM serum concentrations.     

Specific enzyme-multiplied immunoassay and fluorescence polarization immunoassay for cyclosporin compared with Cyclotrac [125I]radioimmunoassay.

The analysis of cyclosporin-A (CsA) has proved a valuable adjunct to clinical care of patients who have received organ grafts. The measurement of CsA in whole blood by specific methods has recently taken a new direction with the introduction of a range of rapid methods, including a homogeneous enzyme immunoassay technique (EMIT) and a monoclonal fluorescence polarization immunoassay (FPIA). The present paper compares these two methods with the established Cyclotrac specific [125I]RIA (radioimmunoassay) using both commercial CsA-spiked control material as well as a group of 60 patient specimens (predominantly renal transplants). While each of the new methods showed acceptable precision and accuracy with the commercial quality control material, significant differences were demonstrated with patient specimens, such that FPIA was 12.5% greater than [125I]RIA (p less than 0.0001), which was in turn 5.9% greater than EMIT (p = 0.007). These data suggested that the FPIA may have residual CsA-metabolite interference and that the EMIT method was the most "specific" for parent CsA of the three tested, potentially therefore more comparable to high-performance liquid chromatography (HPLC).     

Implications of vancomycin degradation products on therapeutic drug monitoring in patients with end-stage renal disease.

In renally impaired patients, vancomycin concentrations typically are maintained at body temperature for extended periods of time due to the drug's prolonged half-life. Both time and increased temperature potentiate production of vancomycin crystalline degradation products (CDP-1). Commercially available vancomycin assays, such as fluorescence polarization immunoassay (FPI) and radioimmunoassay, cross-react with CDP-1 isomers. Overestimation of vancomycin concentrations by 40-53% due to cross-reactivity of CDP-1 with active factor B vancomycin occurs with FPI. As FPI is the most common method of analyzing serum vancomycin, clinicians must be aware of its potential shortcomings and be prepared to alter vancomycin dosages in renally impaired patients. The possibility of adverse affects due to elevated concentrations of CDP-1 or therapeutic failures due to subtherapeutic levels of factor B vancomycin cannot be excluded.     

Quantitation of vancomycin and its crystalline degradation product (CDP-1) in human serum by high performance liquid chromatography

The delayed clearance of vancomycin results in accumulation of vancomycin crystalline degradation product, CDP-1, in the bodies of renally impaired patients. The 2 isomers, CDP-1-M (major) and CDP-1-m (minor), of CDP-1 are antibiotically inactive but cross-react with some immunoassays that use polyclonal antibodies resulting in falsely elevated results. A high performance liquid chromatographic (HPLC) method was developed to quantitate vancomycin and CDP-1 in the serum of renal patients. After solid phase extraction of 200 μl serum, the separation of vancomycin, the 2 isomers of CDP-1 and the internal standard (cefazolin) was accomplished by gradient HPLC on a reversed phase C18 column with detection at 210 nm. Linearity was established from 1 to 25 and 25 to 100 μg ml⁻¹ vancomycin and 1 to 25 μg ml⁻¹ CDP-1. Coefficients of variation for vancomycin and CDP-1 were 3.3–8.6% (n=10) and 2.8–5.2% (n=8).     

Automated fluorescence polarization immunoassay for monitoring vancomycin

We have extended fluorescence polarization immunoassay (FPIA) technology for the measurement of drugs to include the complex amphoteric glycopeptide antibiotic vancomycin (molecular weight, 1,449). Fluorescein-labeled vancomycin was employed as a tracer, and antisera specific for vancomycin were raised in rabbits by conventional procedures. Tracer, sample, and diluted antiserum were combined, and the polarization of tracer fluorescence is determined in a specially designed fluorometer (Abbott TDx). Because of instrument design, the possibility of fluorescent interferences is minimized. The assay can measure as little as 0.6 mg/L of vancomycin and is free of interferences from hemolysis, lipemia, bilirubin, and changes in protein concentration. The coefficient of variation within assay was 3% (n = 5) and between assays was 5% (n = 5). The FPIA assay (TDx Vancomycin) was compared to a liquid chromatographic (LC) assay for vancomycin and to a commercially available radioimmunoassay (RIA) for 98 clinical specimens. A linear least-squares regression analysis gave a correlation coefficient for LC of 0.980 from the equation FPIA = 1.09 LC + 3.04, and a correlation coefficient for RIA of 0.957 from the equation FPIA = 1.036 RIA + 1.66.     

Comparison of bioassay, high-performance liquid chromatography, and fluorescence polarization immunoassay for quantitative determination of vancomycin in serum

This investigation was designed to compare three assay techniques, the traditional bioassay (agar diffusion), and two more recent techniques, high-performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA), for the determination of vancomycin concentrations in serum. One hundred clinical samples obtained from patients receiving vancomycin were assayed by each method. The results from each assay were compared using linear regression analysis. The resultant correlation coefficients were as follows: 0.9996 for the HPLC versus FPIA, 0.7773 for the FPIA versus bioassay, and 0.7779 for HPLC versus bioassay. The FPIA technique was the easiest and fastest of the three methods; FPIA and HPLC were the most accurate.     

Evaluation of an enzyme immunochromatography method for carbamazepine: a comparison with enzyme-multiplied immunoassay technique, fluorescence polarization immunoassay, and high-performance liquid chromatography

A noninstrumented enzyme immunochromatography (EIC) method for monitoring carbamazepine using whole blood was compared to the enzyme-multiplied immunoassay technique (EMIT), fluorescence polarization immunoassay (FPIA), and high-performance liquid chromatography (HPLC). Samples from 74 patients were evaluated in the comparison study, yielding correlation coefficients of 0.961 (EMIT), 0.974 (FPIA), and 0.867 (HPLC). The EIC method produced within-run coefficients of variation of 4.3%, 4.9%, and 5.8% for three carbamazepine concentrations. The between-run coefficient of variation over 107 days was 4.9%. The spiked serum sample analysis yielded recovery rates ranging from 98 to 102%. Enzyme immunochromatography was found to be a useful noninstrumented method for on-site testing. The test gives quantitative patient sample results comparable to the results obtained using established laboratory methods.     

Comparison of nonspecific radioimmunoassay, high-performance liquid chromatography, and fluorescence polarization immunoassay for cyclosporine monitoring in renal transplantation

Three methods, i.e., nonspecific radioimmunoassay (RIA; Incstar), fluorescence polarization immunoassay (FPIA; TDx Abbott), and high-performance liquid chromatography (HPLC), have been used for monitoring cyclosporine blood levels in renal transplantation patients. The levels obtained from 135 samples showed a modest correlation between RIA and HPLC, FPIA and HPLC, RIA and FRIA. The mean ratios of RIA to HPLC, FPIA to HPLC, and RIA to FPIA were 2.96, 4.14, and 0.73. The significant variations in cyclosporine levels result from the cross-reaction of antibody with some cyclosporine metabolites, by which these two methods often overestimate the true blood cyclosporine level. HPLC is a more effective and reliable method for pharmacokinetic studies and blood level monitoring of cyclosporine in clinical practice.     

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.     

Evaluation of fluorescence polarization immunoassay for determination of cyclosporin in plasma

The fluorescence polarization immunoassay (FPIA) method for determination of cyclosporin in plasma was evaluated and compared with the high-performance liquid chromatography (HPLC) and the radioimmunoassay (RIA) methods. The coefficients of variation for the within-run and between-run precision were less than 5 and less than 8%, respectively, for samples ranging in concentration from 50 to 600 ng/ml. Recoveries were determined by adding cyclosporin at concentrations from 25 to 1,000 ng/ml to patient plasma; they were, on average, 98.5%. The calibration curve was stable throughout a 10-week study period. There was no clinically significant interference due to hemolysis, icterus, lipemia, or other commonly used drugs. There was considerable variation of the ratio of the FPIA result to the HPLC result, whereas there was a good correlation between the FPIA and the RIA results (r = 0.975, n = 25, y = 1.2x - 36.4), when evaluated using specimens from renal transplant patients receiving cyclosporin orally. It was concluded that the FPIA is an appropriate, rapid method for patient cyclosporin analysis in plasma and serves as a practical alternative to the RIA.     

高效液相色谱法和荧光偏振免疫法测定万古霉素血清浓度的比较

目的:比较高效液相色谱(HPLC)法与荧光偏振免疫(FPIA)法分别测定血清万古霉素浓度的结果,探讨两者的相关性。方法:收集临床检测万古霉素药物浓度的血清43份,分别用HPLC法和FPIA法进行测定,运用配对t检验,Bland-Altman分析和线性回归分析比较2种方法的测定结果。结果:HPLC法和FPIA法测定的万古霉素血清浓度具有良好的相关性, 回归方程为:Y_FPIA=1.103X_HPLC+0.831 5(R²=0.957 2); Bland-Altman评价分析2种方法一致性较好;配对t检验显示2种方法测定结果之间有显著统计学差异(P<0.000 1)。结论:相较于FPIA法,HPLC法测定不受代谢和降解产物干扰,能准确检测血清万古霉素浓度,适合应用于治疗药物监测。     

Comparison of three commercial amphetamine immunoassays for detection of methamphetamine, methylenedioxyamphetamine, methylenedioxymethamphetamine, and methylenedioxyethylamphetamine

Three commercial immunoassays for detection of amphetamines in urine, Abuscreen radioimmunoassay (RIA), enzyme-multiplied immunoassay technique (EMIT), and the TDx fluorescence polarization immunoassay (FPIA), have been investigated for detection of methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDE). Blank urine was spiked with 0.1 to 3000 micrograms/mL amphetamine analog and used as sample in the assays. With the RIA and FPIA, MDA displayed a higher cross-reactivity to amphetamine than other analogs, but with EMIT, methamphetamine was relatively similar to amphetamine while MDA, MDMA, and MDE were less reactive. The high specificity RIA and the EMIT confirmation reagents for urine amphetamines produced significant, but relatively minor, reduction in the detectability of these analogs. The variation in cross-reactivity seen between the different assays suggests that RIA, EMIT, and FPIA antibodies have different recognition sites; however, all three immunoassays do detect the illicit amphetamine analogs to varying degrees.     

测定环孢素全血浓度的2种方法对比及临床应用

目的对比荧光偏振免疫法(FPIA)和均相酶扩大免疫分析法(EMIT)2种测定方法监测环孢素(CsA)血药浓度的差异。方法收集98例次环孢素全血样品,同日同一患者的全血样品分别采用FPIA和EMIT法测定血药浓度。结果 EMIT和FPIA测定结果相关系数r为0.9795,EMIT法较FPIA法平均低(11±12)%(P<0.05,n=98),其中CsA血药谷值浓度(c_0)EMIT法较FPIA法平均低(14±9)%(P<0.05,n=69)。CsA血药峰值浓度(c_2)EMIT法较FPIA法平均低(2±5)%(P>0.05,n=29)。结论 2种不同方法测定全血中CsA血药浓度具有显著差异,EMIT法较FPIA法具有较高的特异性。     

Methotrexate measurements in plasma: comparison of enzyme multiplied immunoassay technique, TDx fluorescence polarization immunoassay, and high pressure liquid chromatography

One hundred nine patient plasma samples were examined for methotrexate (MTX) levels by enzyme multiplied immunoassay technique (EMIT), fluorescence polarization immunoassay (TDx), and high pressure liquid chromatography (HPLC). EMIT analysis was performed twice within a time span of 18 months. All three methods measure MTX with a high degree of specificity, sensitivity, and precision. There was no evidence of decay of MTX concentrations in samples stored at -20 degrees C for 1.5 years. EMIT, TDx, and HPLC are adequate methods for MTX quantification in the clinical laboratory.     

A modified HPLC method for the determination of vancomycin in plasma and tissues and comparison to FPIA (TDX)

A modified high performance liquid chromatography (HPLC) method for the quantification of vancomycin levels in plasma and tissues is described. The method uses solid phase extraction (SPE) of vancomycin from the samples and reversed phase HPLC with UV detection. The method was fully validated in terms of recovery, linearity, selectivity and various stability conditions. Vancomycin was determined in plasma samples obtained from 15 patients undergoing cardiopulmonary bypass, before and repeatedly during 12 h after drug administration. The vancomycin levels in plasma were measured by HPLC and by fluorescence polarization immunoassay (FPIA) (TDX). The following correlation was found: TDX = 0.84 HPLC + 1.04. The mean vancomycin levels in skin, fat, atrium, pericardium and sternum, before and after bypass, are reported.     

Analytical performance of the CEDIA cyclosporine PLUS whole blood immunoassay

Cyclosporine A (CsA) is a potent immunosuppressive agent used in solid organ and bone marrow transplantation. Because of the narrow therapeutic range and variable pharmacokinetics, blood levels of CsA are routinely monitored. The performance of the CEDIA CsA PLUS whole blood immunoassay was evaluated on the Olympus AU400 trade mark, and results were compared to those obtained by high-performance liquid chromatography (HPLC), enzyme-multiplied immunoassay technique (EMIT), and fluorescence polarization immunoassay (FPIA). A total of 592 whole blood samples from patients receiving CsA were tested by each of the assays. CEDIA was linear from 25 to 2000 micro g/L. Total imprecision ranged from 2.7% to 8.7% at CsA values between 48 and 1502 micro g/L. Recovery of added CsA was within 10% of assigned values and was unaffected by bilirubin and lipemia. Metabolite cross-reactivity at 500 micro g/L was 8.1% for AM1, 21.7% for AM4n, and 32.5% for AM9. Regression analysis revealed the following: HPLC = 0.93. CEDIA - 21.2 (r = 0.975), EMIT = 1.08. CEDIA - 25.2 (r = 0.982), and FPIA = 1.14. CEDIA + 13.4 (r = 0.984). CEDIA has acceptable analytical performance for routine CsA monitoring. Advantages are the absence of an extraction step and an extended measuring range. The disadvantage is the high metabolite cross-reactivity; however, results were similar to EMIT     

Measurement of cyclosporine in plasma of cardiac allograft recipients by fluorescence polarization immunoassay

The Abbott TDx fluorescence polarization immunoassay (FPIA) procedure for measuring cyclosporine A (CsA) was evaluated and compared with the Sandoz polyclonal radioimmunoassay (CsA RIA kit) method. This drug assay was evaluated for precision, calibration, stability, and accuracy. Within-run precision studies utilizing 25 replicate analyses of the three control preparations (containing CsA in the 60-800 ng/ml range) resulted in coefficients of variation (CV) ranging from 1.0 to 9.1%. The CVs of between-run precision determined by assaying the same control drug levels for five consecutive working days ranged from 3.9 to 4.6%. Calibration curve stability was assessed by examining the drift in control values over a 2-week period. Maximum plasma ranged from 82.6 to 108.2%. Four hundred plasma samples were obtained from 30 heart-transplant patients during the first 6 months of CsA therapy and each sample was analyzed simultaneously by TDx and RIA. Linear regression analysis of the results obtained for each patient (x = RIA, y = FPIA) revealed the following mean values: r = 0.87, (CV = 13.7%), slope = 1.47 (CV = 39.2%). Moreover, the concentration of CsA was determined in 35 patient samples both by TDx and high-performance liquid chromatography (HPLC). FPIA results up to 12 times higher than HPLC results have been noted.     

酶扩大免疫测定技术与荧光偏振免疫法测定全血环孢素浓度的比较

目的:比较酶扩大免疫测定技术(EMIT)与荧光偏振免疫法(FPIA)测定全血环孢素浓度的差异。方法:通过分别测定高、中、低浓度标准质控品评价各方法的准确度及精密度,并对卫生部室间质控品及99份临床全血环孢素样本进行测定,比较2种方法测定结果的相关性。结果:EMIT与FPIA方法学的相对回收率分别在93.74%～105.73%与99.84%～102.26%之间,日内及日间精密度良好,RSD均小于10%,符合生物样品的测定要求。两方法测定结果具有良好的相关性Y=1.122 1 X+1.783 7,r=0.987,EMIT的测定结果大多低于FPIA测定结果。结论:EMIT法与FPIA法测定环孢素血药浓度结果存在显著性差异,临床应用中应予以关注并作相应调整。     

The TDx assay for cyclosporine and its metabolites in blood samples compared with HPLC and RIA methods

Two methodologies have been developed to monitor cyclosporine (CsA) therapy: high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). Recently, a fluorescence polarization immunoassay (FPIA) has also become commercially available for the assay of CsA and its metabolites. The authors compared the results obtained with a modified FPIA with those found with two RIAs which use a polyclonal antibody, in order to verify if the FPIA assay is suitable for routine measurements in blood samples. Moreover, the accuracy of the RIAs and of the modified FPIA was checked against the results obtained by an HPLC technique assumed as a reference assay. The FPIA assay for CsA in blood samples seems preferable to the RIAs; in fact, as far as specificity is concerned, the TDx assay is comparable to polyclonal RIAs, while the precision (both within- and between-laboratories) is significantly better. Moreover, the TDx method is easier and faster to perform (20 samples can be assayed in about 30 min, while 2-4 h are necessary with RIA), with fewer handling steps; the instrumentation is automated and the reagents are more stable and less hazardous than those used in RIA.