Relationship between cyclosporine concentrations obtained using the Roche Cobas Integra and Abbott TDx monoclonal immunoassays in pre-dose and two hour post-dose blood samples from kidney transplant recipients

Current evidence suggests that cyclosporine (CsA) concentration in blood samples taken 2 hours after Neoral microemulsion (Novartis Pharmaceuticals; East Hanover, NJ) administration (C2) predicts clinical events in transplant patients better than the pre-dose (trough) concentration (C0). Similarly, previous findings have shown that the metabolites/CsA ratio is substantially lower in C2 than in C0 samples; however the between-monoclonal immunoassay differences for C2 samples have received little attention in the literature. In 56 C samples and 60 C samples from renal transplant patients, CsA levels were determined using the monoclonal fluorescence polarization immunoassay (mFPIA) from Abbott (Abbott Park, IL) and the homogeneous enzyme immunoassay technique (HEIT) from Roche Diagnostics (Basel, Switzerland). In both cases a high correlation coefficient between the results was obtained (r > or = 0.971), with a linear regression for C0 samples: mFPIA = 1.47 HEIT + 22.0 and for C2 samples: mFPIA = 1.11 HEIT + 71.96. The difference between the linear regression slopes was statistically significant (P < 0.001), and the mFPIA/HEIT ratio was significantly higher for C than for C samples (P < 0.001).     

Automated homogeneous immunoassay analysis of cotinine in urine

A study was conducted to evaluate the performance comparison of a homogeneous enzyme immunoassay (EIA) designed to detect cotinine in urine and carbon monoxide (CO) breath measurements to determine smoking status. The clinical comparison was done using urine and breath specimens from 218 volunteers. Urine samples were analyzed by immunoassay and confirmed by gas chromatography-mass spectrometry (GC-MS). Breath carbon monoxide was determined by a commercial analyzer. Using cutoffs of 10 ppm for CO and 500 ng/mL for urinary cotinine, the relative sensitivity/specificity was 93.6%/74.0%. The positive predictive value was 86.8%, and the negative predictive value was 86.5%. However, comparison of the EIA to GC-MS showed a sensitivity/specificity of 96.2%/98.4% and a positive predictive value of 99.3%. The EIA was also evaluated non-clinically for precision, stability, recovery, and interferences. In addition, the non-clinical evaluation demonstrated coefficients of variation from 0.37 to 1.09% across cotinine concentrations ranging from 0 to 5000 ng/mL. The assay was found to be highly specific for cotinine and cross-reacted to a limited degree with 3-hydroxycotinine. Finally, multiple freeze-thaw cycles of urines containing cotinine showed no degradation of the drug in the specimen when tested in the EIA. Thus, the EIA tested is a rapid, lab-based test that can reliably determine cotinine levels and their relation to smoking status.     

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     

Quantitative determination of amorphous cyclosporine in crystalline cyclosporine samples by Fourier transform infrared spectroscopy

The purpose of this study is the development of a quantification method to detect the amount of amorphous cyclosporine using Fourier transform infrared (FTIR) spectroscopy. The mixing of different percentages of crystalline cyclosporine with amorphous cyclosporine was used to obtain a set of standards, composed of cyclosporine samples characterized by different percentages of amorphous cyclosporine. Using a wavelength range of 450-4,000 cm(-1), FTIR spectra were obtained from samples in potassium bromide pellets and then a partial least squares (PLS) model was exploited to correlate the features of the FTIR spectra with the percentage of amorphous cyclosporine in the samples. This model gave a standard error of estimate (SEE) of 0.3562, with an r value of 0.9971 and a standard error of prediction (SEP) of 0.4168, which derives from the cross validation function used to check the precision of the model. Statistical values reveal the applicability of the method to the quantitative determination of amorphous cyclosporine in crystalline cyclosporine samples.     

特异性荧光偏振免疫法监测521例肾移植后环孢素A全血浓度3275次

目的通过监测肾移植后病人环孢素A(CsA)全血浓度 ,提出CsA在三联免疫抑制用药方案中的理想治疗窗。方法用特异性荧光偏振免疫法测定CsA全血浓度 ,对521例病人监测3275次 ,按术后时间及临床表现分组比较。结果肾移植后<1 ,、1～3、3～6、6～12个月、1～2和>2年的CsA全血谷浓度的理想治疗窗应分别为250～450、200～400、150～300、100～250、100～200和100～180μg/L。结论CsA全血浓度在上述范围内 ,中毒反应和排异反应明显减少     

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。     

Monitoring cyclosporin in blood: between-assay differences at trough and 2 hours post-dose (C2)

With the introduction of a cyclosporin monitoring strategy based on the use of a sample collected 2 hours after dosing (C2) rather than the predose sample (C0), there was concern that the differences in blood cyclosporin results from the various assay systems would result in assay-specific target ranges for C2 monitoring. In addition, it was not known if the different proportion of cyclosporin metabolites in the blood 2 hours after dosing compared with that seen in predose samples would alter the relationship between the various assay methodologies. The aim of this study was to address these issues using blood samples from patients who had undergone kidney and liver transplantation. To do this, paired samples were collected predose and 2 hours after cyclosporin dosing at various periods following transplantation in kidney (88 paired samples) and liver (165 paired samples) transplant recipients. Cyclosporin was measured in these samples using five different immunoassays (radioimmunoassay, two fluorescent polarization immunoassays, and two homogeneous immunoassays) and high-performance liquid chromatography-mass spectrometry. The results of the study showed that when using these immunoassays to measure blood cyclosporin concentrations at C0, the cross-reactivity of the antibodies in the different immunoassay kits resulted in target therapeutic ranges that would need to vary between assays to maintain parity. However, when the same assays were used to measure the blood cyclosporin concentration at C2, the results were congruent, and assay-specific target therapeutic ranges should not be necessary. Thus, when adopting a C2 monitoring strategy, it is possible to use target therapeutic ranges that are independent of the assay system used.     

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.     

Comparison of cyclosporine measurement in whole blood by high-performance liquid chromatography, monoclonal fluorescence polarization immunoassay, and monoclonal enzyme-multiplied immunoassay.

Monitoring of cyclosporine concentrations in whole blood is used routinely as a guide to adjusting dose so as to achieve optimal therapeutic benefit with minimal adverse effects. In the present study, we have compared a specific high-performance liquid chromatography (HPLC) assay with a fluorescence polarization immunoassay (TDx) and an enzyme-multiplied immunoassay (Emit). Both Emit and TDx assays employ a monoclonal antibody to cyclosporin A and therefore have the potential for a high degree of specificity. Blood specimens (EDTA as anticoagulant) were obtained from 113 patients (71 renal transplants, 17 liver transplants, and 25 other categories) taking cyclosporine and analysed by all three methods. There were significant correlations between results for HPLC and Emit (Emit = 10.54 + 1.07 x HPLC; r2 = 0.82, p less than 0.001) and between results for HPLC and TDx (TDx = 9.16 + 1.42 x HPLC; r2 = 0.82, p less than 0.001). Compared to HPLC analysis, 74% and 96%, respectively, of Emit and TDx results were to the left of the line of identity. The TDx monoclonal antibody appears to have a lesser degree of specificity than that used in the Emit assay. Mean concentrations of cyclosporine measured by Emit and TDx were 17% and 51% higher, respectively, than those measured by HPLC. Because of this overestimation, we suggest that both Emit and TDx methods may find their most appropriate use in routine therapeutic monitoring of renal transplant patients in whom metabolite concentrations are less variable over time.     

Relative performance of specific and nonspecific fluorescence polarization immunoassay for cyclosporine in transplant patients.

The analytical performance of specific and nonspecific fluorescence polarization immunoassays (FPIAs, Abbott Laboratories) for cyclosporine was compared. Both specific and nonspecific FPIAs demonstrated excellent between-run coefficients of variation (5.9% vs. 3.9%) at three levels of control, and a high degree of between-center reproducibility (r2 greater than 0.96). In addition, the correlation between cyclosporine levels measured by specific and nonspecific FPIAs was statistically significant, though imperfect, in both renal (r2 = 0.70) and cardiac transplant patients (r2 = 0.55). In kidney transplant patients, the nonspecific/specific ratio was significantly higher in patients with serum bilirubin concentration exceeding 3 mg/dl (5.9 +/- 2.6 vs. 2.8 +/- 1.1), due to impaired elimination of cyclosporine metabolites in the bile. The nonspecific/specific ratio was also significantly higher in heart transplant patients early (less than 1 month) posttransplant compared with patients in the late posttransplant period (3.4 +/- 0.8 vs. 2.9 +/- 0.8). The Abbott FPIA provides a highly precise method for measuring cyclosporine, with a turnaround time of 15-20 min. The specific monoclonal FPIA has the additional advantage of measuring primarily unchanged cyclosporine and thus has an imperfect correlation with the nonspecific polyclonal FPIA. Together with clinical data, the use of FPIAs may help to improve the efficiency of cyclosporine therapeutic drug monitoring.     

Validation of a new homogeneous immunoassay for the detection of carisoprodol in urine

The objective of this project was to validate a new high-throughput homogeneous enzyme immunoassay (HEIA) for the rapid detection of carisoprodol in human urine. Carisoprodol (Soma(?)) and meprobamate are widely prescribed as musculoskeletal pain relief drugs and are listed as one of the 10 most frequently identified drugs associated with DUI cases. Carisoprodol has a short elimination half-life of 1-3 h; however, its major active metabolite, meprobamate, has a longer elimination half-life of 6-17 h. As a result, it is important for an immunoassay to cross-react with both compounds. The advantage of this new assay is that cutoff concentrations can be adjusted between 100 and 500 ng/mL. The reportable range was 25 to 1000 ng/mL for carisoprodol and 50 to 10,000 ng/mL for meprobamate. The intraday coefficient of variation (% CV) for the semi-quantitative assay was less than 1%. The homogeneous assay was validated with a total of 86 urine samples previously analyzed by liquid chromatography-tandem mass spectrometry with carisoprodol concentrations ranging from 50 to 10,000 ng/mL. The accuracy was found to be 100% when immunoassay cutoff concentrations of carisoprodol and meprobamate were set at 100 and 1000 ng/mL, respectively.     

单克隆抗体与多克隆抗体荧光偏振免疫分析法测定环孢素 A 全血浓度的比较

本文用单克隆抗体荧光偏振免疫分析法(MAb-FPIA)与多克隆FPIA 法(PC-FPIA)测定肾移植病人环孢素A(CsA)的全血浓度并进行了比较。49例肾移植术后病人口服不同剂量CsA 期间,共监测CsA 全血谷浓度65例次。结果表明。在MAb-FPIA 法观察浓度40～600ng/ml 的范围内,PC-FPIA 法的测定结果为110～1700ng/ml,是MAb-FPIA 法的1.7～3.9倍。两种方法之间有良好的线性关系(r=0.9067,P<0.01)。按术后不同时间分组比较,M/P 比值(MAb-FPIA 法/PC-FPIA 法)随术后时间延长而减小,各组间的差异有显著意义(P<0.05)。     

Monitoring of the free concentration of cyclosporine in plasma in man

Summary The free fraction of cyclosporine A (CsA) and its total plasma concentration as determined by HPLC(CsA_T) were prospectively monitored in 66 recipients of renal transplants. The free CsA levels (CsA_u) were calculated.The variability in free CsA levels was no less than for total CsA_T levels. The correlation between CsA_u and CsA_T was high (r=0.90). Both CsA_T and CsA_u covaried with serum triglycerides and apolipoprotein A1.Fourty-four of the 66 patients suffered acute rejection episodes on 69 occasions. CsA_T and CSA_u both decreased and to a similar extent at the occurrence of acute rejection (42% and 59% decrease, respectively; significant vs baseline. Notsignificant difference in decrease in CsA_TvsCsA_u).Acute nephrotoxicity occurred on 11 occasions in 10 patients. Both CsA_T and CSA_u were approximately twice as high at the time of acute nephrotoxicity as compared to one week previously. Both CsA_T and CsA_u were higher during the first month after transplantation in patients with than in patients without systemic infection.Thus, plasma CsA_u gave no additional clinical information or guidance compared to CsA_T in renal transplant recipients. Due to the complexity of its assay, which requires two consecutive analyses, there does not appear to be any need for routine monitoring of CsA_u in renal transplant recipients.     

Comparison of particle-enhanced turbidimetric inhibition immunoassay and fluorescence polarization immunoassay using monoclonal antibodies for theophylline

The DuPont theophylline assay reagent kit, a particle-enhanced turbidimetric inhibition immunoassay (PETINIA) method adapted for use on a centrifugal fast analyzer, was evaluated. It was compared with fluorescence polarization immunoassay (FPIA). Day-to-day precision was 4.7% at 6.8 micrograms/ml, and 3.3% at 26.6 micrograms/ml. The assay is linear to a concentration of 40 micrograms/ml. Good correlation was found between the two methods (PETINIA/FPIA: y = 1.04x + 0.15, r = 0.988, Syx = 1.19) in the evaluation of 176 patients receiving theophylline. This method offers a precise and accurate alternative to FPIA.     

环孢素血药浓度监测在再生障碍性贫血治疗中的观察

目的探究环孢素A(CsA)浓度监测在再生障碍性贫血(AA)治疗中的临床意义。方法回顾性调查2011年5月至2015年5月经我院确诊为AA的患者48例,对425例次CsA血药浓度监测数据进行统计分析。结果 CsA平均血药浓度为(195.75±162.52)ng/m L,156次监测处于目标血药浓度的范围内(36.7%)。接受首次监测的患者,血药浓度结果在目标范围内16例(33.33%),22例患者的监测结果在目标范围以下(45.83%)。患者血药浓度的监测结果是否处于目标范围与年龄无相关性,差异无统计学意义(P=0.689)。所有患者在治疗期间均接受监测,总有效率为68.75%。此外,接受监测次数小于3次患者的有效率明显低于监测次数大于3次(4~10次)的患者(P=0.002)。结论 CsA的药动学个体差异大,血药浓度监测有助于提高CsA治疗AA的有效率。环孢素浓度监测在再生障碍性贫血治疗中有重要意义,及时准确地监测CSA的血药浓度,可避免或减少治疗中不良反应的发生,提高治疗效果。     

Evaluation of instrumental, nonisotopic immunoassays (fluorescence polarization immunoassay and enzyme-multiplied immunoassay technique) for cyclosporine monitoring in whole blood after kidney and liver transplantation.

Two new instrumental methods, an enzyme-multiplied immunoassay technique (EMIT) and a fluorescence polarization immunoassay (FPIA), were evaluated for monitoring of cyclosporine (CyA) in whole blood samples of renal and liver transplant patients. They are considered as being specific to the parent drug and they were compared with a specific radioimmunoassay (RIA) and a nonspecific FPIA. The data reveal that the novel procedures provide slightly overestimated CyA levels compared with specific RIA (6-12% for EMIT, 20-25% for FPIA). For both assays, intrarun and interrun reproducibilities were found to be in the 2-8% range. The ease of performance and the possibility of performing approximately 40 assays/h make the two methodologies very attractive for both routine and emergency analyses. These approaches are viewed to be complementary to the only previously available instrumental method, the nonspecific FPIA, which provides three- to fourfold higher CyA levels than those obtained with specific methods. Specific and nonspecific monitoring of CyA levels allowed variations in proportions of metabolites to total CyA and metabolites to be distinguished. A higher percentage and variability of cross-reacting metabolites were found in whole blood samples after liver transplantation compared with those in blood of kidney transplant recipients.     

Monitoring of plasma methadone: intercorrelation between immunoassay and gas chromatography-mass spectrometry

Determination of plasma methadone is essential in connection with dose adjustments for patients participating in methadone maintenance programs. We successfully adapted the existing fluorescence polarization immunoassay (FPIA) kit intended for urinary methadone to plasma assays. A concentration interval of 50-900 ng/ml could be covered. The coefficient of variation was less than 7%, and the limit of detection below 50 ng/ml. The intercorrelation between the immunoassay and a specific gas chromatographic-mass spectrometric (GC-MS) method was studied in samples from 19 heroin addicts in methadone maintenance treatment. A total number of 97 plasma samples with a concentration range of 31-842 ng/ml were used. The slope and intercept of the regression line (CFPIA = 0.93 X CGC-MS + 15) was in good agreement with the theoretical relation (CFPIA = CGC-MS), with a coefficient of correlation of 0.978. The mean ratio, in quantitative result, between the techniques (CFPIA/CGC-MS) was 1.03 +/- 0.01 (SEM). We conclude that the immunoassay proposed in this study can be safely used in patients participating in methadone maintenance programs.     

Miniaturization of homogeneous assays using fluorescence polarization

Homogeneous fluorescence-based assays are a significant development in HTS. Fluorescence polarization (FP) is emerging as an important HTS technology that reduces costs while greatly increasing throughput. Inherently suitable for miniaturization, FP has been demonstrated to run in as little as 4 μl total assay volume.     

Comparison of radioimmunoassay with homogeneous enzyme immunoassay for the determination of theophylline concentration in serum.

The homogeneous enzyme immunoassay (EMIT) and radioimmunoassay (RIA) procedures for the quantitation of theophylline concentrations in serum were evaluated and compared. Both methods exhibited curves linear over the therapeutic range (range of concentration, 2.0-33.0 microgram/ml). Precision was acceptable for both methods with the coefficient of variation being less than 8.3%. The RIA procedure was found to be slightly more precise. The correlations between the EMIT and RIA procedures was found to be sufficient to allow the procedures to be used interchangeably. This would facilitate the test availability on a 24 hr, 7 day basis.     

均相酶免疫法与液质联用法测定人血清中卡马西平浓度的比较

目的：比较均相酶免疫法（HEIA）与液质联用法（LC-MS）检测人血清中卡马西平浓度结果。方法：入组110份临床样本,分别用2种方法测定,检验2组检测数据的正态性和显著性差异,采用Spearman法进行相关性分析,绘制Bland-Altman偏差图考察检测方法的差异性,Passing-Bablok回归法考察2种方法等效性。结果：HEIA法和LC-MS法的测定平均值分别为（8.23±5.59）μg·mL^-1和（6.19±4.35）μg·mL^-1。2组数据不呈正态分布,差异有统计学意义（P<0.05）,呈高度相关（r=0.982 0）。2种方法测定值的差值的平均值为（29.5±21.3）%,且不同浓度水平差异相当,一致性较好。Passing-Bablok回归方程为：Y_HEIA=0.012 9+1.318 7X_LC-MS,2种方法不具有等效性。结论：HEIA法比LC-MS法的检测值高,采用2种方法开展卡马西平治疗药物监测工作时,应考虑不同方法的测定差异值,根据临床实际疗效和安全性调整参考范围。