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Technical Briefs |
1 Laboratory of Therapeutic Drug Monitoring, Department of Clinical Chemistry, University Hospital St Luc, University of Louvain, 10 Hippocrate Avenue, B-1200 Brussels, Belgium,
2
Abbott Diagnostics Division, Rue du Bosquet 2, B-1348 Ottignies/Louvain-la-Neuve, Belgium;
a author for correspondence: fax 32 2 764 90 44, e-mail wallemacq{at}lbcm.ucl.ac.be
Cyclosporine (CsA) is a cyclic undecapeptide that has potent immunosuppressive activity, but its narrow therapeutic range and variable pharmacokinetics in humans make monitoring of CsA mandatory (1). The generally accepted whole-blood therapeutic ranges of CsA (with specific assay) are 100–200 µg/L for renal transplant patients and 150–250 µg/L for cardiac, hepatic, and pancreatic transplant patients during the maintenance phase (2)(3), whereas slightly higher ranges are recommended during the induction phase. The CsA metabolites arising from oxidative pathways (4) cross-react in immunoassays (5)(6), leading to results higher than those by HPLC. Trough concentrations of metabolite AM1, the major metabolite in human blood (1)(4), can exceed those of the parent drug (1). For metabolites AM1, AM9, and AM4N, immunosuppressive activity is <10% of that observed for CsA (7)(8), and at least in rats, various CsA metabolites are not nephrotoxic (9). Thus CsA itself, rather than its metabolites, is the major pharmacologically active substance (10), and specific analytical methods are recommended (3)(11)(12). HPLC is specific but time-consuming and labor-intensive; it also displays the highest between-center CV (1). Three major immunoassays based on different CsA monoclonal antibodies were developed by Abbott, IncStar, and Syva Laboratories: a fluorescence polarization immunoassay (FPIA; for the TDx analyzer), a Cyclo-Trac RIA, and an Emit, respectively (13)(14). All of these immunoassays yield slightly higher concentrations than does HPLC, with FPIA concentrations high by 20–30% (15). The results can be summarized as HPLC < Emit < RIA < TDx.
Very recently, a new monoclonal CsA FPIA assay was introduced by Abbott Laboratories for the AxSYM analyzer; it was aimed at reducing metabolite cross-reactivity further and, therefore, at correlating better with HPLC. We first evaluated the analytical performances of this new assay by comparing it with the TDx whole blood monoclonal assay and the Emit assay on Cobas Mira plus. The clinical performance of this AxSYM assay was further evaluated by comparing a total of 150 clinical samples analyzed with the three methods on the same day. In addition, the three methods were evaluated regarding their cross-reactivity with three CsA metabolites. Finally, to evaluate the role of the type of organ transplanted in the correlation AxSYM/TDx, these methods were compared using 608 renal transplant and 112 hepatic transplant samples.
The AxSYM assay requires 184 µL of sample, and 59 samples/h can be
analyzed after a rapid organic extraction (150 µL of whole blood + 50
µL of solubilization reagent + 300 µL of precipitation reagent).
The blood samples after pretreatment were vortex-mixed individually for
10 s and centrifuged 5 min according to the manufacturer's
instructions. The pretreatment volumes are thus similar for the TDx CsA
monoclonal whole blood method (150 µL of whole blood + 50 µL of
solubilization reagent + 300 µL of precipitation reagent, which is a
zinc sulfate solution in methanol and ethylene glycol). For the Emit
method, a pretreatment with methanol and ethylene glycol was used. The
analytical performance of each method is summarized in Table 1
. The within-run imprecision (CV) was determined for the
AxSYM and the comparative methods (TDx and Emit). Each of the three
controls (low, medium, and high) was analyzed in replicates of 20
during 3 consecutive days. In addition, to assess the between-day CV,
each of the three controls was analyzed in replicates of two during 10
different days. The results are shown in Table 1
. To estimate the
detection limit, 10 samples of calibrator A (0 µg/L) were analyzed
with the three methods on 4 different days; the mean + 2 SD was 7.4,
4.2, and 1.5 µg/L, respectively, for AxSYM, TDx, and Emit (Table 1
).
To assess dilution linearity, the AxSYM calibrator F (800 µg/L) was
serially diluted with the AxSYM calibrator A (0 µg/L) to obtain
target concentrations of 800, 400, 200, 100, 50, and 25 µg/L; the
mean results were 800, 399, 196, 94, 45, and 26 µg/L. To determine
whether hepatic or renal impairment interfered, we selected seven
samples with bilirubin values of 242–797 µmol/L and two samples with
creatinine of 496 and 520 µmol/L. These samples were CsA-free. After
addition of known amounts of CsA (300 µg/L), we compared the CsA
concentrations with results in two nondiseased blood specimens
(bilirubin, 9 µmol/L; creatinine, 88 µmol/L). The mean CsA
concentration in the control samples was 292 µg/L, whereas CsA
concentrations were 285–317 µg/L in the cholestatic samples and
299–306 µg/L in the samples from patients with renal
impairment. Neither bilirubin nor creatinine correlated with the
measured CsA concentration.
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We assayed 150 samples (54 from kidney, 35 from hepatic, 29 from heart, and 32 from bone marrow transplantation patients treated with CsA) simultaneously with the three techniques (AxSYM as y-axis and TDx or Emit as x-axis). Deming regression analyses performed with these data (all transplant types together) for AxSYM-TDx yielded the correlation coefficient r = 0.981 and the equation y = (0.78 ± 0.012)x - (3.9 ± 2.16); Sy|x = 11.35; and for AxSYM-Emit, it yielded the correlation coefficient r = 0.974 and the equation y = (0.86 ± 0.015)x + (19.7 ± 2.11); Sy|x = 13.1.
AxSYM results were lower than those from TDx by ~20% and were much
closer to the Emit values (Fig. 1
). A significant decrease in the blood concentrations was
observed vs TDx but not vs Emit.
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To evaluate the cross-reactivity of CsA metabolites in the three
assays, we added 400-1700 µg/L of metabolites AM1, AM9, and AM4N
(provided by Novartis Pharmaceuticals, Basel, Switzerland) to
the 100 µg/L calibrator in all three methods. All measurements were
made in duplicate on the same day. The cross-reactivities for AxSYM
were significantly lower than the values obtained with the TDx and
closer to those obtained by Emit (Table 1
).
In addition, we compared the results obtained for 608 renal and 112 hepatic transplants using the AxSYM with those obtained using the TDx; the least-squares equations were y = (0.81 ± 0.006)x - (2.6 ± 1.03); r = 0.980; Sy|x = 12.2 for renal transplants; and y = (0.71 ± 0.02)x - (3.2 ± 2.1); r = 0.975; Sy|x = 13.3 for hepatic transplants. The slope we observed for the hepatic transplants (0.71) was significantly smaller than those obtained for the total population of transplants (slope = 0.78) and the kidney transplant population (slope = 0.81), which was consistent with the better specificity of the AxSYM method for the CsA parent compound.
The lower results with the AxSYM assay may appear surprising because the AxSYM and TDx assays use the same monoclonal antibody. The increased specificity may arise from the pretreatment and extraction steps. The AxSYM pretreatment solution contains more methanol and less ethylene glycol than that of the TDx. Moreover, the pipetting sequences and incubation times are modified. The FPIA technology is based entirely on the binding constants of the antibody for the analyte. The binding of an antibody to its ligand is affected by several factors, including the concentrations of the protein and ligand, temperature, pH, buffering conditions, and others. In addition, binding is dependent on the length of incubation required to reach equilibrium. The AxSYM method appeared to be reproducible and stable. No calibration was required during the study (>7 weeks), whereas it was necessary to recalibrate the Emit method weekly. The analytical performance (within- and between-run imprecision and detection limit) of the TDx was slightly better. However, it should be stressed that the quality-control samples used in the AxSYM had considerably lower mean values than those used in the TDx. Contrary to the CsA assay on TDx, no special probe cleaning step was required on AxSYM. The concentrations of the AxSYM CsA calibrators were 0, 40, 100, 200, 400, and 800 µg/L, whereas they were 0, 100, 250, 500, 1000, and 1500 for the TDx. Thus, the AxSYM calibrators included more concentrations in the range most frequently encountered in samples.
The major advantage of this new method is better specificity for the parent drug. This improved specificity is demonstrated by the values obtained for clinical specimen compared with the TDx and confirmed by metabolite cross-reactivity, which is ~50% lower than the cross-reactivity observed for TDx. The higher specificity of the new AxSYM method should yield CsA blood concentrations much closer to HPLC data, reflecting more accurately the concentration of the immunosuppressive parent compound. Furthermore, measurement of CsA on a new generation analyzer such as the AxSYM should substantially reduce the laboratory technologist time required for the assay through true random access, shorter time to first result, and the ability of the AxSYM to be bidirectionally connected to a laboratory information system.
Acknowledgments
We want to acknowledge the collaboration with B. Herzig-Moter, Abbott Diagnostics, and to thank Thomas Koshy, Abbott Diagnostics, for the statistical calculations. We also acknowledge the technical skill of C. Luypaert and J. Koenig, and thank Novartis Pharmaceuticals, Basel, Switzerland, for kindly providing the cyclosporine metabolites.
References
The following articles in journals at HighWire Press have cited this article:
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  C. Hartel, N. Schumacher, L. Fricke, B. Ebel, H. Kirchner, and M. Muller-Steinhardt Sensitivity of Whole-Blood T Lymphocytes in Individual Patients to Tacrolimus (FK 506): Impact of Interleukin-2 mRNA Expression as Surrogate Measure of Immunosuppressive Effect Clin. Chem., January 1, 2004; 50(1): 141 - 151. [Abstract] [Full Text] [PDF]
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 C. D. Holt, G. Ingle, and T. M. Sievers Inhibitors of Calcineurin Journal of Pharmacy Practice, December 1, 2003; 16(6): 414 - 433. [Abstract] [PDF]
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