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Analytical Validation of a Homogeneous Immunoassay forDetermination of Mycophenolic Acid in Human Plasma

J.M. Vergara Chozas, A. Sáez-Benito Godino, N. Zopeque García, S. García Pinteño, I. Joumady,C. Carrasco García, and F. Vara Gil

ABSTRACT

Mycophenolic acid (MPA) is an immunosuppression agent for the prophylaxis of organrejection in patients receiving allogeneic transplants. The drug is administered based in 2formulations, mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium(EC-MPS). MPA acts by specific, reversible, uncompetitive inhibition of inosine mono-phosphate dehydrogenase (IMPDH) and thus blocks the proliferation of both T- andB-activated lymphocytes. Therapeutic drug monitoring (TDM) constitutes an importantpart of immunosuppressive treatment because of the demonstrated significant intraindi-vidual and interindividual variability of its pharmacokinetic behavior. TDM is required tooptimize immunosuppressive efficacy. We present the analytical validation of a homoge-neous particle-enhanced turbidimetric inhibition immunoassay (PETINIA) technique fordetermination of MPA in human plasma, and compare with a homogeneous enzymeimmunoassay technique (EMIT; reference method), both methods adapted on a Dimen-sion analyzer (Siemens). We examined 50 human plasma samples from kidney transplantrecipients treated with MMF or EC-MPA, which were analyzed simultaneously by bothmethods. The interassay precision was 5.95% at a concentration of 1.0 �g/mL, 3.47% at 7.5�g/mL, and 3.75% at 12.0 �g/mL. The bias of PETINIA-MPA for each of the 3 qualitycontrol sample was �3.0%. Least squares linear regression yielded an r-value of 0.994 withthe following linear regression equation: PETINIA � 0.939 * EMIT � 0.063. Bland-Altman comparison presented a mean negative difference of �0.312 �g/mL (standarddeviation [SD], 0.441), namely, �7.6% for PETINIA-MPA. The PETINIA assay formonitoring MPA concentrations is an acceptable method for routine clinical use, withinterassay imprecision (% coefficient of variation) ranging from 5.9% to 3.7% below andabove the therapeutic concentration range, respectively. In conclusion, MPA-EMIT andPETINIA-MPA methods on Dimension analyzer have a good correlation (r � 0.994), butPETINIA-MPA method demonstrates a negative average difference of �7.6% in com-

parison with EMIT-MPA method.

ciDBtT

MYCOPHENOLIC acid (MPA) is an immunosuppres-sion agent for the prophylaxis of organ rejection in

atients receiving allogeneic transplants.1,2 The drug isadministered in 2 formulations, mycophenolate mofetil(MMF) and enteric-coated mycophenolate sodium (EC-MPS).3,4

MPA is a potent and specific inhibitor of de novo purinesynthesis by reversible, uncompetitive inhibition of inosinemonophosphate dehydrogenase (IMPDH). The inhibitionof IMPDH in activated lymphocytes by MPA directly

reduces intracellular guanine nucleotide pools. Lympho-

© 2012 by Elsevier Inc. All rights reserved.360 Park Avenue South, New York, NY 10010-1710

Transplantation Proceedings, 44, 2669–2672 (2012)

ytes are very dependent on the availability of sufficientntracellular guanine nucleotide for the production of new

NA, and thus block the proliferation of both T- and-activated lymphocytes.1,5 A recent study suggested addi-

ional mechanisms for MPA-mediated immune suppression.he MPA inhibited T-cell interleukin (IL)-17, interferon

From the Hospital Universitario Puerta del Mar, Cádiz, Spain.Address reprint requests to José Manuel Vergara Chozas,

C/ Gibraltar n° 27, Jerez de la Frontera (Cádiz), Spain, CP:

11407. E-mail: josem.vergara.sspa@juntadeandalucia.es

0041-1345/–see front matterhttp://dx.doi.org/10.1016/j.transproceed.2012.09.063

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(IFN)-�, and tumor necrosis factor (TNF)-� production butot IL-2 production. Phenotypic analysis showed that drugreatment enhanced the expression of negative costimula-ors PD-1, CTLA-4, and the transcription factor FoxP3 andecreased the expression of positive costimulators CD27nd CD28.6

The use of MPA to prevent graft rejection has beenshown in many multicenter clinical trials and has providedopportunities to develop alternative immunosuppressivecombinations.7–9

Therapeutic drug monitoring (TDM) of this agent con-stitutes an important part of immunosuppressive treatment.The drug shows a narrow therapeutic window (1.0 �g/mL to.0 �g/mL), great intraindividual and interindividual phar-

macokinetic variability, and lack of correlation betweenMMF or EC-MPS dose and plasma MPA concentrations.10

The usefulness of monitoring MPA trough level (C0) is beingebated, but other studies suggested its utility in demonstrat-

ng that 12-hour area under the plasma concentration-timeurve (AUC0–12) correlates with both clinical outcomes anddverse effects. It has been proposed as appropriate aherapeutic range for the MPA AUC of 35–75 �g*h/mL,valuated using Enzyme Multiplied Immunoassay Tech-ique (EMIT).11,12 It is recommended to maintain the drug

within the therapeutic window to optimize its efficacy andminimize its side effects.

The aim of this study was to evaluate the analyticalperformance characteristics of an automated immunoassayto quantify MPA in human plasma on the Dimensionclinical chemistry system (Siemens).

METHODS

Fifty whole blood samples were collected from kidney transplantrecipients treated with MMF or EC-MPS, obtained routinely fordiagnostic purpose at different stages after renal transplantation.Blood samples were collected in K2EDTA tubes and centrifugedmmediately at 3000 rpm for 10 minutes; the plasma were analyzedimultaneously using an EMIT 2000 Dimension analyzer (refer-

Table 1. Precision and Accuracy Data for the PETINIAMPA Assay

Control Mean (�g/mL) CV % Bias %

IntraassayLevel 1 1.032 2.18 3.20Level 2 7.510 0.91 0.13Level 3 12.350 3.16 2.91

InterassayLevel 1 1.024 5.95 2.46Level 2 7.536 3.47 0.48Level 3 12.199 3.75 1.66

Abbreviations: Level 1, 1.0 �g/mL; Level 2, 7.5 �g/mL; Level 3, 12.0 �g/mL.

Table 2. Comparison of PETINIA MPA

Slope (95% CI)

PETINIA vs EMIT 0.939 (0.910–0.968) �

Note: Slope, intercept, and correlation coefficient (r) used a least square regressio

nce method) and using a totally automated turbidimetric inhibi-ion immunoassay technique on the Dimension platform.

The EMIT 2000 MPA assay (EMIT) uses a homogeneousnzyme immunoassay technique for the analysis of MPA (referenceethod). It is based on competition for MPA antibody-binding

ites. MPA in the sample competes with MPA labeled with thenzyme glucose-6-phosphate dehydrogenase (G6PDH, from Leu-onostoc mesenteroides). Active (unbound) enzyme converts thexidized nicotinamide adenine dinucleotide (NAD) to NADH,esulting in a kinetic absorbance change that can be measuredpectrophotometrically. Enzyme activity decrease upon binding tohe antibody, allowing the MPA concentration in the sample to beeasured in terms of enzyme activity. The EMIT was performed in

ccordance with the manufacturer’s recommended protocol forimension analyzer.The analytical method studied for MPA in plasma is based on a

omogeneous particle-enhanced turbidimetric inhibition immuno-ssay technique (PETINIA), which uses a synthetic particle-MPAonjugate and monoclonal MPA-specific antibody. MPA present inhe sample competes with MPA on the particles for availablentibody, thereby decreasing the rate of aggregation. Hence, theate of aggregation is inversely proportional to the concentration of

PA in the sample. The rate of aggregation is measured usingichromatic turbidimetric reading at 340 nm and 700 nm. TheETINIA was performed in accordance with the manufacturer’secommended protocol for Dimension analyzer.

To determine repeatability of the MPA PETINIA assay, 3ommercial controls (Mycophenolic Acid Controls, Siemens) con-aining MPA were used. To calculate intra-assay imprecision, eachontrol sample was analyzed 10 times in 1 batch. The inter-assaymprecision was determined analyzing each control sample 1 timever 15 working consecutive days. Accuracy was determined by theeviation from target concentrations for each mean of the 3 qualityontrol interassay samples.

The analytical sensitivity was determined by repeated measure-ent of level 1 calibrator (0 �g/mL) 5 times and adding 2 standard

eviations (SD) to the mean value. The functional sensitivitylowest concentration with a coefficient of variation �20%) wasbtained by dilution 1:1 of level 2 calibrator (0.63 �g/mL) andepeated measurements (10 times). The linearity of dilution wasbtained by dilution of a high concentration sample (13.8 �g/mL)ith another at low concentration (1.0 �g/mL) by dilutions 3:1, 2:2,

and 1:3.Correlation was assessed between both methods (EMIT vs

PETINIA) measuring concentrations of 50 plasma specimenscollected from patients receiving MMF or EC-MPS after renaltransplantation. The statistical analysis of correlation was per-formed using least squares linear regression and Bland-Altmangraph analysis.13 Means of MPA concentrations from both meth-ods were compared using Student t test and significance wasassessed at a P value �.05.

RESULTS

The results of imprecision and accuracy using 3 differentquality controls are presented in Table 1. Intraassay CV%

ension and EMIT Dimension Assays

cept (95% CI) r (95% CI) n

(�0.225–0.098) 0.994 (0.989–0.997) 50

Dim

Inter

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MPA IN HUMAN PLASMA 2671

for PETINIA AMP were 2.18%, 0.91%, and 3.16%. Theinterassay precision values were 5.95%, 3.47%, and 3.75%,for concentrations spanning the calibration range tested.Mean deviations for PETINA AMP from the target con-centration for intraassay and interassay data were 3.2%,0.13%, and 2.91%, and 2.46%, 0.48%, and 1.66%, respec-tively.

The analytical sensitivity was 0.11 �g/mL and the func-ional sensitivity obtained by this assay was at least 0.29

�g/mL (CV, 9.0%).Dilution linearity was between 1.0 �g/mL and 14.0

�g/mL (experimental concentration � [1.006 * theoreticalconcentration] � 0.12).

The results using least squares linear regression analysisare shown in Table 2, demonstrating a strong correlationbetween PETINIA MPA and EMIT MPA methods onDimension, with the correlation coefficient equal to 0.994(95% confidence interval [CI], 0.989–0.996). Figure 1shows the linear regression results of the PETINIA MPAand EMIT MPA comparisons; the slope was 0.939 (95% CI,0.910–0.968) and the y-intercept was �0.063 (95% CI,�0.225–0.098).

Bland-Altman comparison of PETINIA MPA assay toEMIT MPA assay presented a mean negative difference of�0.312 �g/mL (SD, 0.441), namely, �7.6% (Fig 2). Theaverage MPA concentration in renal transplant recipientplasma using EMIT MPA assay was 4.104 �g/mL (range,0.7–20.8) and for PETINIA MPA was 3.792 �g/mL (range,0.6–19.2; P � .001).

DISCUSSION

The role of the MPA assay in the clinical care of patientshas a valuable adjunct to therapeutic and clinical treatment.It has a diagnostic role in distinguishing underdosing fromoverdosing in renal transplant recipients. Two methods aregenerally used to measure MPA in plasma: immunologicmethods and high-performance liquid chromatographic as-says (HPLC). The HPLC methods generally involve the useof expensive equipment and time-consuming sample pre-treatment. The immunologic methods were developed as analternative to the HPLC methods.

The PETINIA assay for monitoring MPA concentrationsis an acceptable method for routine clinical use, with

Fig 1. Correlation of EMIT-MPA vs PETINIA-MPA in plasma of50 patients after kidney transplantation. Yielded r value is 0.994with the following linear regression equation: PETINIA-MPA �

0.939 * EMIT-MPA � 0.063.

nterassay imprecision (%CV) ranging from 5.95% to.75% within and above the therapeutic concentrationange, respectively. The functional sensitivity was at least.29 �g/mL and the linearity over 1.0 to 14.0 �g/mL was

well within acceptable limits. Linearity of dilution is animportant characteristic for the ability to assess out-of-range samples and maintain the accuracy of the finaldetermination. However, the comparisons showed a signif-icant difference between PETINIA and EMIT MPA onDimension analyzer. The degree of mean underestimationby PETINIA was �7.6%, with a concentration range of 0.6to 19.2 �g/mL. The discrepancies between the methodscould be explained by the fact that each assay used differentstandard materials, with different analytical range anddifferent methodological techniques (enzyme immunoassayvs immunoturbidimetric assay) or this bias likely reflectsdifferent cross-reactivity of MPA metabolites to the anti-bodies used in both immunochemical methods.14–16 Fur-ther studies should be performed to determine cross-reactivity of the antibody with MPA metabolites in differenttransplantation populations such as cardiac or liver trans-plants.

Comparison of EMIT MPA versus PETINIA MPAmethods on the Dimension platform with the regressionanalysis showed a good correlation (r � 0.994).

In conclusion, the PETINIA MPA assay run on theDimension analyzer has a good performance as described,allowing precise and accurate measurement of MPA plasmaconcentrations, and is well suited to application in the TDMlaboratory, leading to reduction in the time commitment oflaboratory staff for measurement of MPA plasma concen-trations. This fully automated method on a commonly usedplatform would allow most laboratories to perform thera-peutic MPA monitoring, facilitating rapid dose adjustmentsfor this drug by the clinician.

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