Evaluation of the immunotech cyclosporine direct radioimmunoassay for the determination of whole-blood cyclosporine in renal transplant patients

Therapeutic drug monitoring (TDM) of cyclosporine (CsA) has been an accepted as an essential tool in the management of solid organ transplant recipients. The authors evaluated a new CsA method, Immunotech cyclosporine direct radioimmunoassay (Beckman Coulter, Prague, Czech Republic), for the measurement of whole-blood CsA concentrations. The performance was compared with CEDIA Plus method as well as group mean data for HPLC and other immunoassays available from the International CsA Proficiency Testing Program (www.bioanalytics.co.uk). Regression analysis of patient samples gave a relationship of RIA = 1.0822 CEDIA(+) + 69.84 (r(2) = 0.933). External CsA-spiked proficiency-testing (PT) samples gave a regression equation of RIA = 0.9672 CEDIA(+) + 4.99 (r(2) = 0.996). The correlation with the CEDIA Plus method using patient specimens (hence, including CsA metabolites) suggested that the test RIA method possibly had slightly inferior specificity for parent CsA. The results suggest that the Immunotech cyclosporine direct RIA kit is suitable for the measurement of whole-blood CsA concentrations and maintained clinically acceptable analytic precision and accuracy, displaying CVs of less than 15% and biases of less than 10%. The PT program CsA-metabolite-free samples showed that calibration between methods was comparable with the possible exception of mFPIA/TDx.     

Evaluation of the AxSYM monoclonal cyclosporin assay and comparison with radioimmunoassay

Recently, a semiautomated fluorescence polarization immunoassay (FPIA) for determination of parent cyclosporin (CsA) has been developed for the Abbott AxSYM system. The new CsA assay measures the drug from an extracted whole blood specimen. The authors report here the evaluation of this new assay and the comparison with a previously validated radioimmunoassay (RIA) method (CYCLO-Trac SP). To assess the imprecision, the authors used tri-level controls supplied by both Abbott and Bio-Rad manufacturers. The within-run CV ranged from 4.4% to 7.3% and the between-day CV ranged from 4.4% to 7.6%. Mean recovery of the drug from clinical specimens spiked with kit calibrators was 108.4%. Fluorescence polarization immunoassay AxSYM (y) was correlated to RIA (x) by using 132 trough blood specimens (44 renal, 44 liver, and 44 heart) from transplant recipients and resulted in the following Passing-Bablok linear regression equation: y = 6.7 + 0.97x, r = 0.989, S(x/y) = 12.9. The percentage of overestimation (mean, range) by FPIA AxSYM versus RIA was (3.8%, range -17.7% to 39.1%). The results observed with this new method from follow-up studies in patients during the early course after transplant were not consistently higher than those obtained by RIA. These findings contrast with previously reported results that compared FPIA TDx assay with RIA. The authors conclude that FPIA AxSYM is a precise method for measuring CsA and offers results similar to those obtained by RIA with a marked reduction in assay time.     

Simultaneous estimation of cyclosporin and mycophenolic acid areas under the curve in stable renal transplant patients using a limited sampling strategy

OBJECTIVES: Area under the curve (AUC)-based monitoring of cyclosporin (CsA) could help to optimise therapeutic drug monitoring in certain transplant patients in addition to trough concentration monitoring. It is the method of choice for mycophenolic acid (MPA). The objective of this study was to develop a limited sampling strategy for simultaneous estimation of CsA and MPA AUCs in long-term renal transplant patients. METHODS: Twenty kidney transplant patients treated with CsA and mycophenolate mofetil were included in a pharmacokinetic study more than 6 months after transplantation. Multilinear regression analyses were performed to develop a model enabling the estimation of both drugs' AUCs using a limited number of samples. Dose-normalised data were used throughout the analysis. RESULTS: Trough concentrations of MPA were poorly correlated with AUC, either used alone (r2 = 0.232) or together with other concentrations. Several models for CsA AUC estimation met the predefined criteria (r2>0.9, P<0.05). The AUC of MPA was best estimated by a three-concentration model (AUC=0.58 C20 min+ 0.97 C1 h + 6.64 C3 h + 3.48; r2 = 0.946). These sampling times also applied to CsA AUC (AUC = 1.17 C20 min + 0.68 C1 h + 5.36 C3 h + 4.24; r = 0.985). AUCs estimated using these models in our patients using the jack-knife procedure were found to be precise and unbiased as compared with reference trapezoidal AUCs. CONCLUSION: We were able to develop a multilinear regression model for simultaneous estimation of both CsA and MPA AUCs using only three blood samples taken up to 3 h post-dosing.     

LSS法估算肾移植患者口服环孢素的药时曲线下面积

目的:应用有限采样法(Limited Sampling Strategy,LSS)估算肾移植患者口服环孢素后药时曲线下面积(AUC)。方法:12名肾移植患者单剂量口服CsA微乳制剂,荧光免疫偏振法测定各采样时间点CsA的血药浓度,以多元线性逐步回归法建立数学模型。结果:单个血药浓度-时间点预测AUC0-12的回归模型,线性关系较差,2点和3点预测AUC0-12的回归模型较单点预测好(r2>0．9,P<0．05)。其中C2,C6预测AUC0-12的回归模型(AUC0-12=1017．029 3．047 C2 3.121 C6,r2=0．960, P<0．05)线性关系佳且准确性好。结论:以LSS法估算口服CsA微乳制剂AUC0-12准确性好,临床上可用作CsA治疗药物监测的手段。     

肝、肾移植术后受者环孢素A血药浓度监测的评估

目的探讨肝、肾移植受者环孢素A(CsA)理想的血药浓度监测指标。方法采用荧光偏振免疫法,对65例肝移植受者及136例肾移植受者进行CsA谷浓度(C0)及服药后2 h血药浓度(C2)监测,并对数据进行归纳和分析。结果肝移植受者C2/C0均值为3.56,肾移植受者C2/C0均值为4.8,肝、肾移植受者C2/C0均值有极显著差异(P<0.001);肝移植受者男性CsA血药浓度较女性低。结论C0+C2和C2/C0作为CsA血药浓度监测指标,能更全面地反映CsA体内药物暴露情况和监测CsA肝、肾毒性。     

单克隆荧光偏振免疫法和高效液相色谱法测定环孢素A全血浓度的相关性

目的比较单克隆荧光偏振免疫法（FPIA—m法）和高效液相色谱（HPLC）法测定环孢素A（CsA）全血浓度的相关性。方法分别采用HPLC法和FPIA—m法测定42份肾移植患者的CsA血样，测定值用t检验和线性回归方法进行分析。结果FPIA—m法与HPLC法测定值之间相关性好；回归方程为CF=78．12＋1．21CH，r=0．9893（P〈0．001）。结论FPIA—m法和HPLC法同属于特异性分析法，FPIA—m法测定值均高手HPLC法。     

肝功能对环孢素血药浓度影响的监测

６０位肾移植术后病人的１０８份ＣｓＡ血样用特异性单克隆（ＭＡＦＰＩＡ）和非特异性多克隆荧光免疫偏振分析法（ＰＡＦＰＩＡ）进行测定。结合肝脏功能与术后时间，观察药物浓度的变化。ＭＡＦＰＩＡ和ＰＡＦ－ＰＩＡ两种检测方法之间有线性关系（ｒ＝０．８９２６），二者的测得值具统计学非常显著性差异。肝功能异常的患者ＰＡＦＰＩＡ／ＭＡＦＰＩＡ比值以及回归曲线参数与肝功能正常的患者相比有明显差异，表明肝功能异常的患者ＣｓＡ的代谢产物在体内蓄积而使得检测结果偏高。术后时间对药物浓度变化有一定的作用。术后近期（１０ｄ内）与术后３～７ｗｋ服用等同剂量的ＣｓＡ，血药浓度有明显差异，说明对于肾移植术后服用ＣｓＡ的患者，应持续、定期地进行药物浓度的监测。     

A simple method to calculate cyclosporine dosage to obtain a target C2 drug level

C(2) Cyclosporine (CsA) level, as a surrogate of area under the time-concentration curve (AUC) 0-4 hours, is a good predictor of drug absorption and clinical outcome after kidney transplantation. It has been difficult to define the optimal C(2) level in the individual case and given the broad range of C(2) due to interindividual absorption variability it has been troublesome to determine the drug dose needed to obtain an expected C(2)-CsA concentration. In this study data of 16 stable renal and renal/pancreas recipients treated with prednisone, azathioprine, and CsA (Neoral) managed by C(0) level was examined. CsA concentrations at time 0 (basal), 2, 6, and 12 hours post CsA (Neoral) intake were determined the day of the study. A significant linear regression level was established between C(2) (but not C(0), C(6) and C(12)) and the dosage expressed as mg/kg/d (P = 0.0113, correlation coefficient r = 0.573018). Subsequently, another 27 renal transplant recipients were studied retrospectively and divided into three groups according to posttransplant period: 1 to 6, 7 to 12, and beyond 12 months after transplant. Equations derived from the relationship between C(2) and dose (mg/kg/d) were similar between the three groups and when compared with the first study. A formula obtained from the 27 patients in the whole posttransplant period (mg/kg/d = C(2) x 0.0010208 + 1.86125) was applied to patients of the first study obtaining a regression coefficient between actual and calculated CsA dose of 0.6145 (P = 0.01). A more accurate equation (P = 0.0001, r = 0.5925) was obtained by analyzing 145 C(2) determinations covering a period from 1 month to 8 years following transplant which gave a linear regression line defined by the equation C(2) x 0.001473 + 1.6673. This equation would permit the calculation mg/kg/d of CsA (Neoral) dose to obtain an expected C(2) level. The derived equation shown in this paper has a predictive value of 50% to 60% only, but can help to find adequate dosage in the presence of an inappropriate C(2) level.     

Is the monoclonal fluorescence polarization immunoassay for cyclosporine specific? Comparison with specific radioimmunoassay.

Trough concentrations of cyclosporine (CsA) in whole blood were measured by specific monoclonal 3H-radioimmunoassay (S-RIA) and compared with those obtained by monoclonal fluorescence polarization immunoassay (S-FPIA) in 89 transplant recipients. Differences in the S-RIA:S-FPIA relationship between kidney transplant (KT, n = 59) and heart transplant (HT, n = 24) recipients were investigated. Mean concentrations of CsA were significantly higher by S-FPIA than by S-RIA [191 +/- 127 vs. 166 +/- 124 ng/ml (mean +/- SD), p less than 0.001], with a S-FPIA:S-RIA ratio of 1.25 +/- 0.33. Higher ratios were observed either in patients with low CsA concentrations or in those with a high proportion of metabolites. HT recipients had higher S-FPIA:S-RIA ratios than did KT recipients (1.37 vs. 1.22, t = 1.87, p = 0.065). Within-assay coefficients of variation were lower for S-FPIA than for S-RIA (2.4% vs. 12.3%, p less than 0.001). Monoclonal FPIA is a precise and fast method, more suitable than RIA for therapeutic CsA monitoring in clinical practice. However, our results indicate a 25% higher mean CsA concentration by S-FPIA than by S-RIA (37% in HT recipients), which should be borne in mind until therapeutic and toxic ranges are established or a more specific FPIA method is developed.     

Comparison of a monoclonal antibody fluorescent polarization immunoassay with monoclonal antibody radioimmunoassay for cyclosporin determination in whole blood.

We have compared the whole blood concentrations of parent cyclosporin A (CsA) using monoclonal fluorescence polarization immunoassay (FPIA) and radioimmunoassay (RIA) as well as polyclonal FPIA in kidney, heart, and bone marrow transplant patients (n = 89). A good correlation was found between monoclonal FPIA and monoclonal RIA (r = 0.96) and a slightly worse one between polyclonal and monoclonal FPIA (r = 0.90). The interassay coefficient of variation was satisfactory for all the methods-less than 5% for monoclonal FPIA. The monoclonal FPIA assay with Abbott TDx appears to provide rapid, precise, and accurate measurement of parent CsA. It is therefore useful for therapeutic monitoring of CsA in whole blood in kidney, heart, and bone marrow transplant patients.     

Measurement of cyclosporine A in rat tissues and human kidney transplant biopsies--a method suitable for small (<1 mg) samples

Therapeutic drug monitoring is used to individualize cyclosporine A (CsA) dosing after transplantation. However, immunosuppressant concentrations within the graft may better predict clinical outcomes, including toxicity. This study aimed to develop a method suitable for CsA measurement using routine fine-needle biopsy samples. CsA was quantified retrospectively in kidney and liver tissues from 10 rats administered CsA, and 21 core needle kidney biopsies taken from renal transplant patients with suspected graft dysfunction. Dried biopsies were weighed (mean ± SD weights of 0.22 ± 0.18 mg), enzymatically solubilized, and then CsA was extracted and quantified using online 2-dimensional liquid chromatography-tandem mass spectrometry. The method was linear (r2 > 0.997, n = 10), accurate, and precise (quality control and calibrator coefficient of variation and bias <15%), with minimal matrix effects (coefficient of variation and bias <15%). Reproducibility of tissue weight measurements was confirmed by retrospective DNA quantitation, with a significant linear correlation between weight and total DNA concentration (r2 = 0.988). In rats, there was a significant linear correlation between CsA concentrations in liver and kidney tissues (r2 = 0.996) but there was no correlation between blood (C0) and tissue CsA concentrations (Spearman r = 0.430 and 0.503, P > 0.05). Similarly, in 16 transplant patients, for whom blood CsA concentrations (C2) were available within 1 day of the renal biopsy being performed, there was no significant correlation between CsA concentrations in blood and kidney tissue (Spearman r = 0.168, P > 0.05). In situ CsA measurements acquired using this method could make an easy transition into clinical use due to their retrospective nature and minimal disruption to current clinical protocols and could provide an additional tool for optimizing clinical outcomes in the future.     

HPLC—MS法和mFPIA法测定全血环孢霉素A及其代谢物的比较

目的:对测定全血环孢霉素 A 的 HPLC-MS 法和单克隆荧光偏振免疫分析法(mFPIA 法)进行评价。方法:建立HPLC-MS 测定全血环孢霉素 A 及其代谢物的方法,采用多元线性回归对 HPLC-MS 法和 mFPIA 法测定的结果进行比较。结果:当患者肝、肾功能异常时,mFPIA 法测定结果同环孢霉素 A 及主要代谢物浓度呈线性相关,易导致检测结果偏高。结论:当患者肝、肾功能异常时,mFPIA 法不适宜作为环孢霉素 A 的临床监测方法。     

肾移植患者口服国产环孢素A软胶囊后治疗药物监测指标的选择

目的:探讨肾移植患者口服国产环孢素A(CsA)软胶囊后治疗药物监测的适宜指标。方法:采集10例肾移植术后稳定期患者口服国产CsA软胶囊后12h内不同时间点的血样,以单克隆荧光偏振免疫(mFPIA)法测定血药浓度。计算各采样点CsA浓度与AUC0～12和AUC0～4的相关系数,考察相关系数间的差异性。结果:除0.5、0.75h外,其余各时间点与0h的相关系数两两间检验未显示有统计学意义的差异(P>0.05)。结论:对于肾移植术后稳定期口服国产CsA软胶囊患者,谷浓度(c0)仍然可以作为适宜的治疗药物监测指标之一。     

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.     

环孢霉素A在服药患者血浆中的分布

目的了解环孢霉素A在服药患者血浆中的分布。方法检测26例肾移植患者CsA全血及血浆谷值浓度和24例。肾移植患者CsA全血及血浆峰值浓度。结果26例。肾移植患者CsA全血及血浆谷值浓度分别为（84．89±43．04）μg·L-1、（15．46±7．34）μg·L-1,24例肾移植患者CsA全血及血浆峰值浓度分别为（606．63±167．10）μg·L-1、（188．39±105．86）μg·L-1。结论血液中的CsA主要同血细胞结合,随着血药浓度的降低,血浆中药物分布的比例也随着减少。     

Considerable lack of agreement between S-FPIA and EMIT cyclosporine assay in therapeutic drug monitoring, of heart transplant recipients

The authors performed a comparative analysis of 60 whole blood samples containing cyclosporine (CsA) from heart transplant (HTx) recipients (n = 60) by the two "specific" monoclonal immunoassays, enzyme-multiplied immunoassay technique (EMIT) and fluorescence polarization immunoassay (S-FPIA), using the Altman-Bland approach based on graphical techniques and simple calculations. The CsA blood concentrations measured by S-FPIA [mean (SD): 268.1 (108.8) ng/mL] showed a statistically significant difference (P < 0.001) from the corresponding concentrations measured by EMIT [219.6 (118.7) ng/mL]. The CsA concentrations were 27% (median) higher when determined by monoclonal S-FPIA than by EMIT. The comparison between EMIT and S-FPIA showed a good correlation (S-FPIA conc. (ng/mL) = EMIT conc. (ng/mL) x 0.88 + 76.1, r = 0.96, P < 0.001). However, a high correlation does not mean that the two methods agree, and their use as interchangeable might be misleading. The authors summarized the degree of agreement by calculating the bias estimated by the mean difference (d) and the standard deviation of the difference (SD). For CsA concentration data, the mean difference (S-FPIA minus EMIT) is +49.9 ng/mL and SD is 31.2 ng/mL. Altman-Bland analysis indicates considerable lack of agreement between EMIT and S-FPIA, with discrepancies of more than 100 ng/mL. The present study's data clearly show that there is a considerable and clinically unacceptable lack of agreement between the S-FPIA and the EMIT techniques in HTx recipients for the whole range of concentrations evaluated (25-500 ng/mL), and this is caused by the variation in the overestimation of the CsA parent compound. Even though a similar CsA reference range was reported during maintenance therapy for both methods (150-250 ng/mL), which might encourage their interchangeability in the clinical setting, this approach should be avoided. Laboratory reports should always state both the concentration of CsA and the analytical method.     

Lack of apparent effect of assay methodology on the pharmacokinetics of digoxin

The purpose of this study was to determine if serum digoxin concentration data using three different automated immunoassay methods would produce similar pharmacokinetic values in normal volunteer subjects. Area under the curve (AUC), steady-state volume of distribution/bioavailability ratio (Vd/F), terminal elimination rate constant (beta), clearance/bioavailability ratio (CL/F), maximum digoxin concentration (Cmax), minimum digoxin concentration (Cmin), and time of peak (Tp) were evaluated. Ten healthy volunteers received digoxin capsules 0.2 mg daily for 10 days. On day 10, 16 serial blood samples were collected over a 24-h dosing interval and analyzed by radioimmunoassay (RIA) (Concept 4, Micromedic Systems), fluorescence polarization immunoassay (FPIA) (TDx, Abbott Laboratories), and affinity column-mediated immunoassay (ACMIA), (aca, duPont Instruments). When comparing RIA and FPIA, the mean of the percent differences for AUC, Vd/F, beta, and CL/F were 9, 4, 10, and 6%, respectively. The mean of the percent differences were 2, 3, 44, and 6%, respectively, when comparing RIA and ACMIA. However, none of these differences were statistically significant. Although a trend toward higher Cmax values by RIA was noted, there was no statistical difference in Cmax, Cmin, and Tp. Orthogonal regression of all serum digoxin concentrations showed that FPIA = 0.76 RIA + 0.19, r = 0.967 (p less than 0.001); and ACMIA = 0.92 RIA + 0.04, r = 0.943 (p less than 0.001). At serum digoxin concentrations less than 1 ng/ml, FPIA overestimated RIA results (p less than 0.005), while ACMIA was approximately equal to the RIA results.(ABSTRACT TRUNCATED AT 250 WORDS)     

LC-MS/MS法和mFPIA法测定全血环孢素A浓度的相关性比较

目的:对高效液相色谱电喷雾串联质谱(LC-MS/MS)法和单克隆荧光偏振免疫(mFPIA)法测定肾移植术后稳定期患者不同时间点的全血环孢素A(CsA)浓度进行相关性比较。方法:分别采用LC-MS/MS法和mFPIA法测定患者全血CsA浓度,对测定结果进行双侧配对t检验比较差异性,用Passing-Bablok回归分析法比较不同采血时间点2种方法测定结果的相关性。结果:2种方法测定值之间具有显著性差异;服药后0、3.0、4.0h时2法测定值的相关性最好,0.5、0.75、12.0h相关性最差。结论:对CsA血药浓度监测结果的分析需考虑监测方法以及不同采血时间点的影响。     

Cyclosporin A absorption profiles in pediatric renal transplant recipients predict the risk of acute rejection

The current focus of cyclosporin A (CsA) monitoring in adult transplantation for optimized immunosuppression is on the early portion of the CsA area under the concentration-time curve (AUC), particularly in the first 4 hours postdose, designated as AUC(0-4), and on the blood concentration 2 hours postdose (C2) as a highly predictive marker for AUC(0-4). Because data in pediatric patients are scarce, full-time (12 hours) and absorption profiles of CsA were analyzed in relation to CsA effectiveness in 61 pediatric renal transplant recipients aged 3.2 to 17.4 years on an immunosuppressive triple regimen with CsA, mycophenolate mofetil, and methylprednisolone. CsA dosing was based on body surface area and adjusted to CsA trough levels. Pharmacokinetic (PK) profiles were obtained 1 and 3 weeks (initial period) and 3 and 6 months posttransplant (stable period). Patients with an AUC(0-4) < 4400 microg x h/L at both PK sampling periods in the first 3 weeks posttransplant had an adjusted relative risk of 48.4% to suffer an acute rejection episode (ARE), whereas in patients with at least 1 AUC0-4 above this threshold, the adjusted relative risk for an ARE was only 13.1% (P < 0.02). The single PK parameters C0 or C2 did not discriminate between patients with and without acute rejection. The PK parameters C1.25 (r2 = 0.64) or C2 (r2 = 0.60) showed a stronger relationship to the absorption profile (AUC(0-4)) than C0 (r2 = 0.15). An abbreviated profile consisting of the PK variables C(0.5;2) or C(0;0.5;2) showed the closest correlation to the absorption profile (r2 = 0.89) and the lowest percentage prediction error. These data indicate that absorption profiling in pediatric renal transplant recipients has the potential to optimize immunosuppressive therapy with CsA.