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Letters |
Institute of Liver Studies, King's College Hospital, and School of Medicine, Denmark Hill, London SE5 9RS, UK
a Address correspondence to this author at: Institute of Liver Studies, Guy's, King's, St. Thomas' School of Medicine at Denmark Hill, Bessemer Road, London SE5 9PJ, UK. Fax 44-171-346-3760; e-mail michael.tredger{at}kcl.ac.uk
To The Editor:
Recent correspondence to Clinical Chemistry (1)(2) addressed the performance of the second generation Tacrolimus assay for the IMx analyzer (Abbott Diagnostics), with the former letter identifying nonequivalence in results from its predecessor and the latter considering performance approaching the lower limits of detection. Our own published results (3) have considered these points, and we report here our additional experience.
In their comparison of the second- vs the first-generation assays, Garg
et al. (1) described comparable coefficients of variation
(CVs), but this is not the case when identical control samples are used
in each assay at low concentrations (
5 µg/L), e.g., 14.2% vs
42.4% at 4.2 µg of tacrolimus per liter of blood (1). In
common with previous findings (3)(4)(5), Garg et al.
(1) reported lower values with the second-generation assay
using 36 samples of undefined origin. The slopes and intercepts
reported for these various comparisons differed (as did the comparison
methods applied), but Garg et al. (1) did not relate these
data into the practical measurement of mean differences in assay
results. Mean underestimates of 1–2 µg/L were reported for
tacrolimus concentrations of 3–35 µg/L both by Wallemacq et al.
(4), who used renal and liver recipients, and ourselves
(3) (adult and pediatric liver and adult renal transplant
recipients and patients with autoimmune disease). Of the explanations
proposed for these differences by the various investigators, the lower
recovery of the second-generation assay experienced by Garg et al.
(1) in 1998 is not consistent with 1998 data from the
Tacrolimus International Proficiency Testing Scheme (coordinator, Dr.
D.W. Holt, St George's Hospital Medical School, London, UK),
from which can be calculated a positive bias and mean recovery
of 114% (range, 102.1–121.7%) in 20 samples to which 3–28 µg/L
tacrolimus was added and a similar overestimate relative to the results
reported by the small number of centers using HPLC/mass spectrometry. A
bias in assay calibrators (3) or differences in the
contribution of tacrolimus metabolites (3)(4)
may be a more likely explanation for the differences between the first-
and second-generation assay results. Given the inherent variability in
assay performance and tacrolimus pharmacokinetics, we still doubt
whether a difference of 1–2 µg/L in assay results would have
major practical impact on management by the realistic clinician.
This is true both early after transplantation, when tacrolimus trough
concentrations usually exceed 10 µg/L (but are subject to variability
because of alterations in graft function, drug dosage, and
coadministered medication), and later in clinically stable patients,
when tacrolimus concentrations are often below 10 µg/L (and
pharmacokinetic variability is lower but still subject to the influence
of food intake) (6). In this lower range, where the
increased sensitivity of the second-generation assay is advantageous,
the CVs in tacrolimus measurements will span the differences of 1–2
µg/L between assay results.
Applying the concept of functional sensitivity (analyte concentration
at 20% interassay CV) to the second-generation tacrolimus assay,
Schambeck et al. (2) have defined a value of 3.1
µg/L for single measurements and recommend the use of two replicates
at such concentrations. However, it is difficult to justify duplicate
measurements realistically in terms of cost-benefit, the inherent
variability in biological determinants of drug concentrations referred
to above, or the use of concentrations as an adjunct to indicators of
graft function and clinical condition in regulating dosage. The
functional sensitivity also may not necessarily equate with the
practical lower limit of quantification of the assay in routine use,
particularly because two mode 1 calibrators (tacrolimus-free samples)
are used to adjust the calibration curve in every assay. Thus, in
defining a practical lower limit for routine assay of tacrolimus, it
could be argued that intraassay variability (i.e., in relation to the
mode 1 or tacrolimus-free calibrators run on the same carousel) may be
of greater relevance than the corresponding interassay variability used
in determining functional sensitivity. Within an assay, and like
Schambeck et al. (2), we reported that tacrolimus
concentrations of 1–2 µg/L blood are distinguishable from
tacrolimus-free samples at high significance and without overlap
(3). Moreover, recent data from the Tacrolimus International
Proficiency Testing Scheme (coordinator, Dr. D.W. Holt) reported a CV
of 17.1% on a sample containing 3.0 µg/L circulated to 184 centers
and a CV of 21.7% on a corresponding sample of 2.0 µg/L analyzed at
172 centers. In addition to reporting a minimum detection limit of
~1.5 µg/L (3), our own routine experience using low
concentrations of tacrolimus has been acquired from 179 interassay
measurements of control material containing 3 µg/L (obtained over 11
months with multiple operators and calibration curves). Despite a CV of
30.9%, acceptable results ranged from 1.1 to 4.5 µg/L when limits of
3 SD were used (outside which we routinely and immediately recalibrate
the assay). Our additional experience with the second-generation assay
over 28 months and >9500 samples is that results 
2.0 µg/L have
been obtained on known tacrolimus-free blood samples on only three
occasions. This concentration (2 µg/L) is used as our minimum
quantifiable concentration.
References
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