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Technical Briefs |
1
Clin. Chem.,
2
Kidney Transplant., and
3
Hepatic Transplant., University Hosp. St Luc, Univ. of Louvain, 10 Hippocrate Ave., B-1200 Brussels, Belgium;
a address for correspondence: Dept. of Clin. Chem., Lab. of Therapeutic Drug Monit., University Hosp. St Luc-U.C.L., 10 Hippocrate Ave., B-1200 Brussels, Belgium, fax +32-2-764-37-32, e-mail wallemacq{at}lbcm.ucl.ac.be
In 1992, an automated whole-blood microparticle enzyme immunoassay was developed (Abbott Labs.) for the measurement of tacrolimus concentrations (TAC I) on the IMx analyzer (1). This method involves the antitacrolimus monoclonal antibody developed by Fujisawa Pharmaceutical, the company producing the immunosuppressant tacrolimus (2). The assay requires 100 µL of whole blood, and 24 samples can be analyzed in ~40 min after a rapid organic extraction (200 µL of precipitation reagent: ZnSO4 solution in methanol and ethylene glycol). The capture reagent consists of latex microparticles to which tacrolimus antibodies are attached, the enzyme is tacrolimus-conjugated alkaline phosphatase, and the substrate is 4-methylumbelliferyl phosphate. This method yields CVs of ~10% (11.8% and 9.6% at concentrations of 15 and 25 µg/L, respectively) (1), but is limited by the detection limit of 5 µg/L. Because in current clinical practice a nonnegligible percentage of transplant patients display low tacrolimus concentrations (<6 µg/L), the immunoassay has recently been modified. A new assay, IMx tacrolimus II (TAC II), with a lower detection limit, has been developed, requiring 150 µL of whole blood and 150 µL of precipitation reagent. Whereas the TAC I has a dynamic range of quantification from 5 to 60 µg/L, the TAC II assay has a range from 1 to 30 µg/L, better corresponding to the therapeutic range of tacrolimus (5–15 µg/L) (3). Because this new assay will replace the TAC I, we have evaluated and compared the two assays in terms of their analytical performances, and their correlation in clinical blood specimens obtained from kidney and liver transplant patients.
Analytical performances were evaluated on the same IMx analyzer, by the same technician, and on the same days. The pipettes used were calibrated before the study. All the analyses (mode 1 calibrator, controls, and patients' samples) in the study were run in duplicate, and blood samples after pretreatment were vortex-mixed individually for 10 s according to the manufacturer's instructions.
Each of the three controls [target concentrations low (L), medium (M),
and high (H): 15, 25, and 40 µg/L, respectively, for the TAC I; and
5, 11, and 22 µg/L for the TAC II)] was used in replicates of 20
during three consecutive days. The within-run precision overall results
were similar for the two assays (Table 1
). Mean values for the TAC I controls were 14.3, 23.9, and 38
µg/L with CVs of 6.2%, 5.7%, and 5.2%, respectively. The
corresponding values for the new assay were 5.4, 12, and 23.7 µg/L
with CVs of 7.1%, 6.7%, and 6.7%. In addition, to assess the
between-run precision, each of the three controls was analyzed in
replicates of two during 10 consecutive days. Mean values for the TAC I
assay were 13.7, 23.7, and 40.5 µg/L with CVs of 7.3%, 5.1%, and
7.1%, respectively. Comparative results for TAC II were 4.9, 10.7, and
23.1 µg/L with CVs of 10.0%, 5.0%, and 7.2%, respectively. The
detection limit of both assays has been evaluated by two approaches:
first, by testing 10 samples of calibrator A (0 µg/L), and second, by
testing 10 tacrolimus-free clinical specimens (transplant patients
under another immunosuppressive scheme). The limits of detection of TAC
I, defined as the lowest concentrations that can be distinguished from
zero with 95% confidence (mean + 2 SD), were 3.8 and 6.4 µg/L for
the first and second approaches, respectively, and the corresponding
results for the TAC II assay were 0.8 and 1.16 µg/L, respectively.
These results confirm the detection limit of ~5 µg/L for the TAC I
assay as described by Abbott, and suggest for the new assay a detection
limit of ~1 µg/L. To compare the two calibration curves, TAC I
controls L and M (15 and 25 µg/L) have been measured in triplicate on
the TAC II assay, resulting in similar mean concentrations (14.2 and
25.4 µg/L, respectively).
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Correlation between the TAC I assay and the new TAC II assay in
clinical practice has been evaluated with 70 whole-blood EDTA specimens
obtained from kidney and hepatic transplant patients treated with
tacrolimus. All samples were analyzed with quality-control material
within a week. Samples with concentrations <5 µg/L (n = 9) have
been excluded from the comparison study. With TAC I and TAC II data
reported in the x- and y-axes respectively, the
correlation coefficient was 0.960 and the regression equation was
y = 0.81x + 0.50. Thus TAC II results were
lower than those from TAC I by 9–17% in concentrations of 5–30
µg/L (according to the equation). To illustrate this bias, a
Bland–Altman plot for method comparison is shown in Fig. 1
(4). In this graph, the differences between the
assays are plotted against the averages of the two methods. Horizontal
lines represent the mean difference (1.26 µg/L) and mean difference
plus or minus 2 SD (mean ± 2.63 µg/L).
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Analysis of the data based on the organ transplanted (n = 30 for liver, n = 31 for kidney) produced no significant changes in the correlation coefficient. The slope for the hepatic transplant samples was 0.73 and the y-axis intercept was 1.0, but the difference was not statistically significant. The positive intercept value compensates partly for the low slope, resulting in similar concentrations in the lower range. During these clinical sample analyses, three control samples were simultaneously added. Mean (n = 10) low, medium, and high concentrations were: 14.0, 25.3, 42.0 mg/L and 5.2, 11.3, 24.3 mg/L for TAC I and TAC II, respectively, similar to the values obtained in the precision study, thus confirming the absence of shift in the curves over time. In addition, three blood specimens (A16, B16, C16) from the tacrolimus European quality-assessment scheme organized by D. Holt, London, UK, were analyzed by both techniques. A16 and C16 are aliquots of tacrolimus-free blood to which 20 and 40 µg/L tacrolimus have been added, and B16 is an aliquot of a single pool of blood samples from transplant patients, presenting a consensus mean of tacrolimus concentration of 13.8 µg/L. The results obtained in our laboratory with both assays are: 23.1, 47.8, and 14.8 µg/L for TAC I, and 19.3, >30 (36.9 after dilution), and 11.6 µg/L for TAC II, respectively.
The reliability of the new TAC II assay is based on good reproducibility (similar within-run and between-run CVs to TAC I), obtained in a concentration range much lower than previously possible. This improvement clearly better corresponds to the current dosage regimen of tacrolimus. The new assay has a detection limit of 1 µg/L, which is similar to that obtained from ELISA [3, 5]. In addition, during our evaluation, the new assay presented an excellent stability of the calibration curve. The first calibration of TAC II obtained in September 1996 was still valid 4 months later, whereas the TAC I assay was recalibrated two times during the same period of time. The observation of tacrolimus concentrations significantly lower with the new assay appears slightly surprising since both assays use the same monoclonal antibody. A possible explanation could be the different volume of blood and precipitation reagent involved in the assays. Some coprecipitation or drug trapping in the protein clot might occur in the TAC II, mainly with fresh blood samples and less with pretreated and hemolyzed samples as controls. This would be consistent with the need of agitation duration of at least 10 s for TAC II (150 µL of blood + 150 µL of precipitation reagent) to avoid incomplete breakup of the erythrocytes. Such need was less critical for TAC I (100 µL of blood + 200 µL of precipitation reagent). Owing to the higher proportion of solvent used in the TAC I assay, a higher extraction ratio of some tacrolimus metabolites could also contribute to a higher result as compared with the new assay. This would also explain why control samples produce data closer to expected values and to TAC I values than do patients' samples. Nevertheless, taking into consideration the clinical practice of tacrolimus therapeutic monitoring, the new IMx tacrolimus II assay, with the improved sensitivity, should meet the need of most users.
Acknowledgments
We acknowledge the collaboration with C. Lhoëst and B. Herzig-Moter from Abbott Diagnostics, and the technical skill of C. Luypaert.
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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Z. T. Cao, M. W. Linder, A. W. Jevans, G. Brown, and R. Valdes Jr Comparison of Tacrolimus Concentrations Measured by the IMx Tacrolimus II vs the PRO-TRAC II FK506 ELISA Assays Clin. Chem., October 1, 1999; 45(10): 1868 - 1870. [Full Text] [PDF]
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J. M. Tredger, C. D. Gilkes, and C. E. Gonde Performance of the IMx Tacrolimus II Assay and Practical Limits of Detection Clin. Chem., October 1, 1999; 45(10): 1881 - 1882. [Full Text] [PDF]
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C. M. Schambeck, A. Bedel, and F. Keller Limit of Quantification (Functional Sensitivity) of the New IMx Tacrolimus II Microparticle Enzyme Immunoassay Clin. Chem., October 1, 1998; 44(10): 2217 - 2217. [Full Text] [PDF]
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U. C. Garg, G. Austin, C. Barnes, and M. Hamilton Comparison of the Abbott IMx Tacrolimus I and Tacrolimus II Assays Clin. Chem., August 1, 1998; 44(8): 1783 - 1785. [Full Text] [PDF]
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