Antibody-conjugated Magnetic Immunoassay Research Articles

Sensitive, wide-range and high-throughput quantification of cyclosporine in whole blood using ultra-performance liquid chromatography coupled to tandem mass spectrometry and comparison with an antibody-conjugated magnetic immunoassay.

Because either trough or peak concentration at 2 h after administration is measured in routine therapeutic drug monitoring for cyclosporine A (CyA), a quantification method with a wide-range calibration curve capable of simultaneously measuring both concentrations is required. We developed a sensitive, wide-range and high-throughput quantification method for CyA in whole blood using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and compared patients' blood CyA levels measured by UPLC-MS/MS and antibody-conjugated magnetic immunoassay (ACMIA). Whole blood samples were prepared by solid-phase extraction using Oasis HLB μElution plate. The UPLC-MS/MS assay showed excellent linearity over a wide calibration range of 5-2500 ng/mL. Within-batch accuracy and precision as well as batch-to-batch accuracy and precision fulfilled the criteria of US Food and Drug Administration guidelines. The blood CyA concentrations measured by the UPLC-MS/MS assay correlated strongly with those measured by ACMIA. A Bland-Altman plot showed a fixed error between CyA concentrations measured by the two methods, and the concentrations measured by the UPLC-MS/MS method were consistently lower than those measured by ACMIA. We have succeeded to develop a sensitive, wide-range and high-throughput quantification method for CyA in whole blood using UPLC-MS/MS.

6 Immunological aspects of ovarian follicle ovulation and corpus luteum formation in cattle

Ovarian follicle ovulation and subsequent luteinization have been described as a controlled inflammatory event, comprising tissue damage and repair. To elaborate this further in cattle, the contribution of immune cells to dominant follicle luteinization, ovulation, and corpus luteum formation was investigated. Ovulation in beef heifers was synchronized using an 8-day progesterone-based synchronization program. Heifers were slaughtered at a local abattoir at 5 timepoints (T): (T1) 24h before ovulation (n=10); (T2) 2h before ovulation (n=9); (T3) 6h after ovulation (n=10); (T4) 24h after ovulation (n=10); (T5) 72h after ovulation (n=10), and ovarian tissue was collected and returned to the laboratory on ice. Follicular fluid, theca, granulosa, and corpus luteum (CL) tissues were recovered by dissection and processed for analysis. The concentrations of a panel of cytokines were measured using an antibody-conjugated magnetic bead immunoassay. The abundance of T-lymphocytes, mast cells, neutrophils, eosinophils, monocytes, macrophages, and dendritic cells was determined by immunohistochemistry. The mRNA relative abundance of candidate genes, including angiogenic growth factors, adhesion factors, chemokines and cytokines, was determined by quantitative real-time PCR analysis. The resulting datasets were analysed using the linear mixed model procedure of SAS and data are presented as least squares means; reported differences were deemed significant at P≤0.05. The cytokines IFNy, IP-10, IL-10, IL-36RA, MCP-1, MIP-1a, MIP-1b, and VEGF-A were detected in follicular fluid. The concentrations (pg mL−1) of IL-10 and VEGF-A were significantly higher in T1 follicular fluid samples compared with T2 (7.70 vs. 0.86 and 2193.33 vs. 293.93, respectively). Although dendritic cells were the most abundant cells in bovine ovulatory follicular and early corpus luteum tissue at all time points (P<0.05), their numbers peaked in ovulatory (T2) thecal tissue (261.5 cells/mm2). The greatest number of neutrophils was identified in thecal tissue at T1 (45/mm2); thereafter, their numbers declined to 1.1/mm2 in CL tissue by T5. Similarly, the numbers of T-lymphocytes, mast cells, monocytes, and macrophages declined in CL tissue at T4 and T5. Candidate gene mRNA expression profiles appeared to be time- and tissue-specific; for example, IFNA was highest in the preovulatory granulosa tissue (T1), IL8 was highest in peri-ovulatory thecal tissue (T2), VEGFA and MMP9 were highest in the early CL tissue (T4 and T5), MMP1, TIMP1, and VCAM1 expression was highest in theca, granulosa, and CL tissue collected on or after ovulation (T2, T4, T5), expression of the prostaglandin-related genes PTGES and PTGS2 was lowest in CL tissue, and that of PTGIS was highest. The current findings support the hypothesis that ovulation in heifers is characterised by an initial proinflammatory cascade followed by a dramatic switch to tissue repair, growth, and remodelling, all occurring within a 72-h period and commencing with the LH surge. Our results highlight the roles of neutrophils, dendritic cells, and macrophages as the key actors in this process.

Pharmacodynamic Monitoring of Tacrolimus-Based Immunosuppression in CD14+ Monocytes After Kidney Transplantation.

Monocytes significantly contribute to ischemia-reperfusion injury and allograft rejection after kidney transplantation. However, the knowledge about the effects of immunosuppressive drugs on monocyte activation is limited. Conventional pharmacokinetic methods for immunosuppressive drug monitoring are not cell type-specific. In this study, phosphorylation of 3 signaling proteins was measured to determine the pharmacodynamic effects of immunosuppression on monocyte activation in kidney transplant patients. Blood samples from 20 kidney transplant recipients were monitored before and during the first year after transplantation. All patients received induction therapy with basiliximab, followed by tacrolimus (TAC), mycophenolate mofetil, and prednisolone maintenance therapy. TAC whole-blood predose concentrations were determined using an antibody-conjugated magnetic immunoassay. Samples were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and phosphorylation of p38MAPK, ERK, and Akt in CD14 monocytes was quantified by phospho-specific flow cytometry. Phosphorylation of p38MAPK and Akt in monocytes of immunosuppressed recipients was lower after 360 days compared with before transplantation in the unstimulated samples [mean reduction in median fluorescence intensity 36%; range -28% to 77% for p-p38MAPK and 20%; range -22% to 53% for p-Akt; P < 0.05]. P-ERK was only decreased at day 4 after transplantation (mean inhibition 23%; range -52% to 73%; P < 0.05). At day 4, when the highest whole-blood predose TAC concentrations were measured, p-p38MAPK and p-Akt, but not p-ERK, correlated inversely with TAC (rs = -0.65; P = 0.01 and rs = -0.58; P = 0.03, respectively). Immunosuppressive drug combination therapy partially inhibits monocyte activation pathways after kidney transplantation. This inhibition can be determined by phospho-specific flow cytometry, which enables the assessment of the pharmacodynamic effects of immunosuppressive drugs in a cell type-specific manner.

Characterization of circulating antibodies with affinity to an epitope used in antibody-conjugated magnetic immunoassays from a case of falsely elevated cyclosporine A

BackgroundWe report a case demonstrating falsely elevated cyclosporine A (CsA) levels by using the antibody-conjugated magnetic immunoassay (ACMIA). To determine the cause of the false positive result, we examined the presence of an interferent in the patient's sample. MethodsA 26-year-old male patient displayed a high CsA concentration eight weeks after discontinuation of CsA treatment when measured by ACMIA. The possibility of assay interference was examined using serial dilution tests, heterophilic blocking tubes, erythrocyte washing, polyethylene glycol (PEG) protein precipitation, and a Protein A sepharose bead immunoabsorption assay. ResultsThe serial dilution test showed nonlinearity, suggesting the presence of an interferent. CsA concentrations in the patient's plasma pre- and post-heterophilic blocking tube treatment were similar. Erythrocyte washing and PEG protein precipitation tests indicated the existence of a protein interferent in the plasma. Moreover, the Protein A bead immunoabsorption assay identified the interferent as antibodies against a unique epitope on the monoclonal antibody-β-galactosidase (mAb-β-gal) enzyme conjugate used in CsA ACMIA. ConclusionsWe identified a circulating antibody against the unique epitope of the CsA ACMIA mAb-β-gal conjugate that induces falsely high CsA concentrations. These findings suggest that clinically aberrant CsA levels require immediate re-measurement by reference methods such as mass spectrometry.

False Increase in Whole Blood Tacrolimus Levels due to Interference in an Antibody-Conjugated Magnetic Immunoassay Method

False Increase in Whole Blood Tacrolimus Levels due to Interference in an Antibody-Conjugated Magnetic Immunoassay Method

Multicenter Analytical Evaluation of the Automated Electrochemiluminescence Immunoassay for Cyclosporine

Cyclosporine A (CsA) is used as a posttransplantation immunosuppressant drug, and careful monitoring of CsA concentration in whole blood is essential. A new automated electrochemiluminescence immunoassay (ECLIA) for CsA measurement has been assessed in a multicenter evaluation. Residual EDTA whole blood samples from patients undergoing CsA therapy after organ transplant were used in assay evaluation at 5 clinical laboratories in Europe. Experiments included imprecision according to CLSI EP5-A2 (within-run and intermediate), lower limit of quantification, linearity according to CLSI EP6-A, and recovery of commercial external quality control samples. In addition, comparisons to liquid chromatography-tandem mass spectrometry methods in routine use at each investigational site and to commercial chemiluminescent microparticle immunoassay and antibody-conjugated magnetic immunoassay methods were performed. Imprecision testing gave coefficients of variation of less than 9% in the 30-2000 mcg/L range for both within-run and intermediate imprecision. Lower limit of quantification of 6.8 mcg/L at one investigational site and 1.8 mcg/L at a second site at 20% coefficient of variation were observed. Linearity was measured over the concentration range 0-2000 mcg/L, yielding a deviation of less than ±12%. External quality control sample recovery by ECLIA was 93%-114% of LC-MS/MS sample recovery. Deming regression analysis of ECLIA method comparison to combined LC-MS/MS results yielded a slope of 1.04 [95% confidence interval (CI), 1.03-1.06] and intercept of 2.8 mcg/L (95% CI, 1.5-4.1 mcg/L). Comparison to chemiluminescent microparticle immunoassay yielded a slope of 0.87 (95% CI, 0.85-0.89) and intercept of 1.4 mcg/L (95% CI, -0.89 to 3.7 mcg/L); comparison to antibody-conjugated magnetic immunoassay yielded a slope of 0.96 (95% CI, 0.93-0.98) and intercept of -4.2 mcg/L (95% CI, -7.1 to -1.2 mcg/L). The data from this multicenter evaluation indicate that the new ECLIA-based cyclosporine assay is fit for its purpose, the therapeutic monitoring of CsA.

Comparison of Antibody-Conjugated Magnetic Immunoassay and Liquid Chromatography-Tandem Mass Spectrometry for the Measurement of Cyclosporine and Tacrolimus in Whole Blood

The aim of this work is to compare the results of a commercially available liquid chromatography tandem mass spectrometry (LC-MS/MS) method in a clinical pathology laboratory for routine Therapeutic Drug Monitoring (TDM) of cyclosporine (CsA) and tacrolimus (Tacr) in pediatric patients with those obtained with the current antibody-conjugated magnetic immunoassay (ACMIA). Whole blood levels of CsA (n= 135) and Tacr (n=100) were sequentially analyzed by using ACMIA and LC-MS/MS on pediatric transplanted patients. The differences were analyzed by using the Passing Bablok regression analysis and the Bland and Altman test. The LC-MS/MS method showed excellent reproducibility and lower limits of quantification compared to the ACMIA. A linear relationship between ACMIA and LC-MS/MS was obtained for both CsA Tacr. No significant inter-method biases were observed. The analytical performances of the LC-MS/MS method make it suitable for the accurate measurement of CsA and Tacr in pediatric transplanted patients. However ACMIA results are also accurate and reliable. For this reason the choice of the method to be used in a routine clinical pathology laboratory can be made on the bases of non-analytical considerations such as costs, organization, availability of skilled personnel.

Follow-Up of Tacrolimus Breastfed Babies

Tacrolimus is a calcineurin inhibitor immunosuppressant approved in the prevention of organ allograft rejection or in the treatment of acute graft rejection. The human experience of tacrolimus during pregnancy is reassuring with no specific pattern of neonatal complications, except possible transient hyperkalemia (1). Although the available data on human milk transfer indicate that only very limited amounts of tacrolimus are excreted into breast milk (2), clinical data on the safety of breast-feeding for the newborn of tacrolimus-treated patients are still very scarce. As a member of the European Network of Teratogen Information Services, our center provides advice on the safety and risk of medications for pregnant and lactating women. We report our experience regarding the safety of tacrolimus for newborns whose mother opted for breast-feeding after delivery. Since 2003, we have received advice queries concerning 14 women treated with tacrolimus. After individual assessment including the maternal treatment, dose, comedications, indication, characteristics of the neonate, and the expected type of breast-feeding (exclusive or not), clinical and biologic follow-up of the infant was systematically proposed if the mother decided to breast-feed. After oral consent, the clinical follow-up consisted of a standardized questionnaire filled during telephone interviews of the patient during and after the period of breast-feeding. To monitor the infant’s exposure to tacrolimus through breast-feeding, measurement of whole-blood tacrolimus concentration was also proposed within 2 to 3 weeks after starting breast-feeding to avoid any residual presence of tacrolimus in the newborn caused by transplacental passage and tacrolimus long half-life (up to 43 hr in healthy adults). No immunologic monitoring was proposed. Among these 14 patients, breast-feeding was initiated in 9, but 3 discontinued within 5 to 15 days (2 because of breast-feeding difficulties and 1 because of mycophenolate mofetil initiation), and they were not included in the follow-up. Data from Table 1 provide details on the mothers, the infant’s characteristics at birth, medication exposure, and follow-up of children for the remaining evaluable six patients. All six women (mean age, 32 years) had received tacrolimus throughout pregnancy for the prophylaxis of solid organ rejection. The mean daily dose was 9.6 mg/d (4.5–15 mg/d). Concomitant drugs included azathioprine in four women, diltiazem in one, and prednisolone and low-dose aspirin in one. The mean gestational age at delivery was 37.1 weeks, and four of six newborns were moderately premature (34–36 weeks). Breast-feeding was exclusive in four patients and partial in two. The median duration of breast-feeding was 3 months (range, 1.5–6 months), and the median duration of infant follow-up was 8.5 months (range, 2–30 months). Whole-blood tacrolimus concentration measured in four neonates between day 15 and day 27 after delivery were undetectable (lower limit of detection <1.9 μg/L, antibody-conjugated magnetic immunoassay; Siemens Healthcare Diagnostics, Tarrytown, NY). Clinical follow-up of the infants did not identify any adverse outcome that might be related to tacrolimus maternal treatment, and developmental milestones were within the expected ranges. In particular, no specific pattern of infection was observed. A longer follow-up of these infants is expected.TABLE 1: Details on mothers and child exposureOnly four previous reports have provided data on tacrolimus exposure of infants through breast-feeding. Jain et al. (2) measured tacrolimus concentrations in 10 colostrum samples from six mothers treated with tacrolimus during pregnancy. The highest tacrolimus concentration was 1.9 μg/L, corresponding to a maximal dose of 0.29 μg/kg per day delivered from milk to the neonate, that is, 0.1% of the lowest pediatric dose (based on an infant milk ingestion of 150mL/kg/d). None of these mothers had breast-fed. The very low tacrolimus exposure of infants during breast-feeding was documented by two other reports that included the measurement of tacrolimus levels in mature milk and, respectively, found a mean tacrolimus concentration of 0.6 and 1.8 μg/L in milk (3, 4). Chusney et al. (5) showed a similarly quick postpartum decrease in tacrolimus serum levels in neonates exposed throughout pregnancy, whether or not subsequently breast-fed, suggesting higher exposure in utero than through human milk. Finally, only one report included the clinical follow-up of an exclusively breast-fed neonate with normal physical and neurologic development at 2.5 months of age (3). To the best of our knowledge, the present study is the first one to provide relatively long-term follow-up in infants exposed to tacrolimus through breast-feeding. These results confirm previous analytical data suggesting that tacrolimus exposure in breast-fed babies is negligible and probably safe even in case of prematurity, therefore allowing the mother to breast-feed. Until more data are available, particularly regarding long-term safety, careful infant monitoring is required, especially in patients on a multiple immunosuppressant regimen. Aurore Gouraud Nathalie Bernard Anne Millaret Méri Bruel Nathalie Paret Jacques Descotes Thierry Vial Poison Center and Pharmacovigilance Department Lyon University Hospitals Lyon, France

Falsely Abnormally Elevated Blood Trough Concentration of Tacrolimus Measured by Antibody-Conjugated Magnetic Immunoassay in a Renal Transplant Recipient: A Case Report

This report presents a falsely abnormally elevated blood trough concentration (Ct) of tacrolimus measured by antibody-conjugated magnetic immunoassay (ACMIA) methods in a renal transplant recipient. Because the Ct of tacrolimus was 78.5 ng/mL at day 2 after a 52-year-old man underwent renal transplantation, we stopped the tacrolimus extended-release formulation. However, because the abnormally elevated blood Ct continued in the range of 41.1–59.1 ng/mL, we then measured the tacrolimus concentration in a stored blood sample before renal transplantation, it was 43 ng/mL. Consequently, the day-7 blood sample was measured with both ACMIA and enzyme-linked immunoassay, showing Ct values of 42.8 ng/mL and 0.89 ng/mL, respectively. Because the abnormally elevated Ct was falsely measured by the ACMIA method, we restarted tacrolimus However, the calcineurin inhibitor was subsequently converted to cyclosporine at day 21 after renal transplantation. Although cyclosporine was also measured by ACMIA, there was not an abnormally elevated Ct. Subsequently, the tacrolimus concentration ratio in plasma and whole blood (P/B-tacrolimus concentration ratio) was measured by ACMIA in a posttacrolimus blood sample. The P/B-tacrolimus concentration ratio was 100%. In contrast, the P/B-tacrolimus concentration ratio was <30% in 2 control patients administered tacrolimus. It has been reported recently that there were cases showing falsely slightly elevated Ct of tacrolimus within the therapeutic range of concentrations. Therefore, we must be careful not to reduce the tacrolimus dose falsely. We consider confirmatory methods for a falsely abnormally elevated Ct of tacrolimus measured by ACMIA to (1) measure P/B-tacrolimus concentration ratio, (2) compare ACMIA with another measurement, and (3) evaluate a blood sample stored before tacrolimus administration.

Increased Cyclosporine Concentrations in the Absence of Cyclosporine Administration

A 9-year-old girl was admitted to our hospital with juvenile metachromatic leukodystrophy (arylsulfatase A deficiency). Symptoms of this lysosomal storage disease, such as decreased school performance and compromised motor skills, had started 1 year earlier. She underwent bone marrow transplantation from a matched unrelated donor after receiving conditioning with fludarabine, treosulfan, and thiotepa, together with thymoglobulin, a rabbit antithymocyte globulin. Conditioning was well tolerated, and the posttransplantation period was uneventful except for 1 febrile episode. Graft-vs-host disease (GvHD)4 prophylaxis consisted of 3 methotrexate doses in combination with a starting daily cyclosporin A (CsA) dosage of 3 mg · kg−1 · day−1. Trough CsA concentrations were monitored with the antibody-conjugated magnetic immunoassay (ACMIA) for CsA (RxL Dimension; Siemens). CsA concentrations entered the therapeutic interval after 3 days, and the dosage was adjusted to achieve trough concentrations of 120–150 μg/L (Fig. 1). The patient received CsA for 16 weeks. During this period, the CsA concentration was measured 31 times, with results from 98 μg/L to 219 μg/L (mean, 156 μg/L). Four weeks after transplantation, the patient developed a mild GvHD of the skin, which disappeared immediately after commencement of prednisolone treatment. Because B lymphocytes were absent or low early after transplantation, the patient received immunoglobulins for 5 months (Fig. 1). Endogenous immunoglobulin production started only in the later phase of immune reconstitution. Six weeks after discontinuation of CsA therapy, a concentration of 147 μg/L was still detected in whole blood. Although the patient was not supposed to have received CsA and the parents had denied the administration of CsA or drugs other than those prescribed, CsA concentrations between 116 μg/L and 174 μg/L were obtained over the next 4 months. Fig. 1. Time courses of the whole-blood CsA concentration measured with the ACMIA CsA assay, the administered daily CsA dose, and the B-lymphocyte count. ### QUESTIONS TO CONSIDER 1. Has the CsA therapy really been discontinued? 2. Can delayed drug elimination explain the continued increased CsA concentrations? 3. What …

Clinically unexpected cyclosporine levels using the ACMIA method on the RXL dimension analyser

Safe use of cyclosporine (CsA) in solid organ transplantation relies on regular whole-blood drug monitoring. Several promising immmunoassays, e.g. the antibody-conjugated magnetic immunoassay (ACMIA) method, were developed and commercialized during recent years to compete with liquid chromatography coupled to tandem mass spectrometry, which remains the reference method but is labor-intensive. We describe the occurrence of interference in the monitoring of whole-blood CsA after transplantation when using the ACMIA method and discuss the potential mechanisms involved in such interference. Clinically unexpected results of whole-blood CsA require immediate reassessment by another technique to prevent the risk of CsA underdosage and graft rejection.

Falsely elevated tacrolimus concentrations measured using the ACMIA method due to circulating endogenous antibodies in a kidney transplant recipient

Background Therapeutic monitoring of whole-blood concentration of tacrolimus, a potent immunosuppressive drug used after organ transplantation, is essential to avoid toxic effects and to maintain the correct dosage. Although the reference method for the determination of tacrolimus concentrations is LC-MS/MS, several certified immunoassays are widely used for routine examinations. We report falsely elevated blood tacrolimus concentrations using the antibody-conjugated magnetic immunoassay (ACMIA) from Siemens Healthcare Diagnostics for the analysis of a patient who had undergone renal transplantation. Methods Whole-blood samples from a patient with elevated tacrolimus concentrations not consistent with the clinical picture were analysed with an alternative immunoassay and were investigated for interference by performing double dilution tests, by incubating in heterophilic blocking tubes and by evaluating plasmatic interfering factors. Results Double dilution tests showed a clear nonlinearity, suggesting that antibody interference not related to heterophilic antibodies had occurred; false-positive concentrations of cyclosporin obtained when using an antibody-conjugated to β-galactosidase suggested the presence of endogenous antibodies directed against β-galactosidase. Conclusion In patients receiving tacrolimus, continued surveillance by laboratory staff and clinicians is necessary when using a method not requiring external pre-treatment, such as the Siemens ACMIA method.

Apparent Elevation of Cyclosporine Whole Blood Concentrations in a Renal Allograft Recipient

We describe a 77-year-old cyclosporine-treated renal allograft recipient in whom falsely elevated whole blood cyclosporine concentrations were encountered using the antibody conjugated magnetic immunoassay (ACMIA) for therapeutic drug monitoring.

Evaluation of the Architect ® tacrolimus assay in kidney, liver, and heart transplant recipients

The narrow therapeutic range of tacrolimus requires therapeutic drug monitoring to prevent transplant rejection and to minimize nephrotoxicity. The aim of this study was to evaluate the analytical performance of the tacrolimus chemiluminescent microparticle immunoassay (CMIA) in everyday practice comparatively with other methods. CMIA imprecision and accuracy were tested using low, medium, and high concentrations in control samples. The limits of quantification (LOQ) of CMIA and antibody-conjugated magnetic immunoassay (ACMIA) were evaluated using negative whole-blood samples containing 0.4–5.7 ng/ml of tacrolimus from a stock solution. CMIA was compared with ACMIA, enzyme multiplied immunoassay (EMIT), and liquid chromatography–tandem mass spectrometry (LC–MS/MS), using 176 samples from recipients (135 men and 41 women) of heart ( n = 19), kidney ( n = 107), or liver ( n = 50) transplants. CMIA total precision was 5.7%, 3.7% and 3.6% with the low-, medium-, and high-concentration controls, respectively; corresponding values for accuracy were 98%, 104%, and 104%. LOQ was 0.5 (95%CI, 0.22–1.38) with CMIA and 2.5 ng/ml with ACMIA. Linear regression results were as follows: CMIA = 1.2LC–MS/MS + 0.14 ( r = 0.98); CMIA = 0.93EMIT + 0.36 ( r = 0.975); CMIA = 1.15ACMIA − 0.25 ( r = 0.988); and, for tacrolimus concentrations in the 1–15 ng/ml range, of special interest as many transplant recipients are given low-dose tacrolimus, CMIA = 1.05LC–MS/MS + 0.38 ( r = 0.94). Two patients had falsely elevated tacrolimus concentrations due to interference in the ACMIA assay; one was a renal transplant recipient who stopped her treatment and had tacrolimus concentrations of 12.5 ng/ml by ACMIA and <0.5 ng/ml by CMIA; the other was an HIV-positive renal transplant recipient whose tacrolimus concentrations by ACMIA were 1.8–43.7-fold those by CMIA. Such interferences with ACMIA, which may be related to endogenous antibodies in the plasma, are likely to negatively impact patient care. In conclusion, the tacrolimus CMIA assay is suitable for routine laboratory use and does not suffer from the interferences seen with ACMIA in some patients.

Rheumatoid Factor Interference in a Tacrolimus Immunoassay

Recently, there has been an interest in the use of tacrolimus for the treatment of rheumatoid arthritis (RA). The role of rheumatoid factor (RF) as a cause of immunoassay interferences is well known. This study is the first to investigate the susceptibility of a tacrolimus immunoassay to interference by RF. Tacrolimus apparent concentrations were determined using the antibody conjugated magnetic immunoassay (ACMIA) run on the Dimension RxL Immunoassay System in 100 randomly selected samples previously submitted for routine diagnostic or monitoring of RA in patients not receiving tacrolimus. Fifty of them had an RF concentration exceeding 100 IU/L and 50 had an RF concentration below 20 IU/L. Samples with tacrolimus apparent whole-blood concentrations above 2.3 ng/mL (limit of quantification of the ACMIA assay alleged by the vendor) were considered as potential false positives. No positive tacrolimus result was found among the 50 samples with serum RF < 20 IU/mL. Among the 50 selected samples from patients with RF > 100 IU/mL (RF range 110-2650 IU/mL), 2 were positive for tacrolimus with ACMIA. In both cases, the pretreatment of these samples with an immunoglobulin blocking agent reduced the apparent tacrolimus concentrations to below the limit of detection. This was confirmed using the alternative and reference tacrolimus assays, both of which reported results below their respective limits of detection. The measured human anti-mouse antibodies levels were found to be elevated. These results show that certain patients with positive RF can have false-positive tacrolimus results using the tacrolimus ACMIA-Flex immunoassay on a Dimension RXL analyzer, which was not the case with 2 other techniques. The interference with the tacrolimus ACMIA results was suppressed after preincubation with an immunoglobulin blocking reagent.

Falsely elevated whole-blood tacrolimus concentrations in a kidney-transplant patient: potential hazards

Tacrolimus-based immunosuppression is the most frequently prescribed immunosuppression for kidney-transplant (KT) patients. Because tacrolimus has a narrow therapeutic window, drug monitoring is mandatory. Of the many methods used to assess whole-blood trough levels, antibody-conjugated magnetic immunoassay (ACMIA) is popular because, compared with microparticle enzyme-linked immunoassays (MEIA), there is no need to pretreat samples, thus reducing time taken by the laboratory technician. Herein, we report on a KT tacrolimus-treated patient who experienced falsely elevated whole-blood tacrolimus concentrations after using the ACMIA method. ACMIA gave trough levels of 24 ng/ml, whereas the actual trough level, when measured by enzyme-multiplied immunoassay technique (EMIT) and high-performance liquid chromatography coupled with mass spectrometry (LC-MS/MS), was nil. After a workup we only found one factor that might have caused the elevated concentration: positive anti-double stranded DNA autoantibodies. We conclude that, when ACMIA produces surprisingly high tacrolimus concentrations in organ-transplant patients, these should be reassessed immediately using either LC-MS/MS or another immunoassay in order to eliminate falsely elevated results.

Comparison of Blood Tacrolimus Concentrations in Liver and Kidney Transplant Recipients using ACMIA and MEIA Immunoassays

Background: Published data on the performance of the new Dade Behring antibody conjugated magnetic immunoassay (ACMIA) for tacrolimus determination are scarce. The aim of this study was to compare the results obtained using the ACMIA and Abbott microparticle enzyme immunoassay (MEIA), which is the most widely used method for therapeutic tacrolimus monitoring.Methods: Trough tacrolimus concentrations were determined in 305 blood samples from kidney (n=138) and liver (n=167) transplant recipients using the ACMIA and MEIA immunoassays. The MEIA results were corrected for hematocrit values lesser than 30% and higher than 40% (Hermida et al. Clin Lab 2005; 51: 43-45).Results: The obtained ACMIA within- and between-run variation coefficients (<10.8%) were acceptable. In the comparison between ACMIA and MEIA results in the blood samples studied, the regression equation was: ACMIA=1.02MEIA+0.29 (r=0.912, p<0.001), with an acceptable difference between the means (8.13±0.53 ng/mL vs. 7.62±0.50 ng/mL). However, in accordance with the well-established interference of the hematocrit on the MEIA results, a highly significant negative correlation between the MEIA/ACMIA ratio and the hematocrit values was obtained (r=-0.585, p<0.001). When the MEIA results were corrected according to the hematocrit (MEIAHtC), the regression with ACMIA levels was: ACMIA=1.08MEIAHtC-0.09 (r=0.926, p<0.001). This equation was analogous to that obtained between ACMIA and MEIA tacrolimus concentrations in the 164 blood samples with hematocrit of 30-40%.Conclusions: ACMIA is an acceptable option for therapeutic tacrolimus monitoring, with an important decrease in technician time in relation to the widely used MEIA.