Limit of Quantification (Functional Sensitivity) of the New IMx Tacrolimus II Microparticle Enzyme Immunoassay

Central Laboratory, Medical Department, University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany
^a Author for correspondence. Fax 0049-931-2012793; e-mail c.schambeck{at}medizin.uni-wuerzburg.de.

To the Editor:

In kidney and liver organ transplant recipients, tacrolimus concentrations of 5.0–15.0 µg/L and 3.0–8.0 µg/L, respectively, should be achieved during long-term course.

Measuring with the old IMx® tacrolimus assay, the laboratory could not provide tacrolimus concentrations in the subtherapeutic range because of its poor sensitivity. The new IMx Tacrolimus II microparticle enzyme immunoassay (Abbott) was introduced in clinical practice 1 year ago. The imprecision and detection limit of this new assay have already been evaluated and compared with the old assay. Wallemacq et al. (1) reported in the October 1997 issue of Clinical Chemistry that the IMx Tacrolimus II assay is characterized by a much better analytical performance.

The detection limit of this new assay was reported to be 1.2 µg/L or 1.5 µg/L (1)(2). Thus far, the interassay imprecision of the new IMx tacrolimus assay has been determined with the help of kit controls. The concentration of the low control (5 µg/L) is within the therapeutic range of tacrolimus. The authors did not publish data on the imprecision of the assay in the subtherapeutic range (1)(2)(3).

Hence, how precise are concentrations measured by the new assay in the subtherapeutic range? This question remains unanswered because the detection limit, defined as the lowest concentration that can be distinguished from zero, insufficiently reflects the performance of an assay in the subtherapeutic range. The analytical detection limit, as defined above, does not represent a lowest concentration that can be measured with a defined imprecision in the long-term course. Spencer (4) proposed the concept of "functional sensitivity", which is defined as the lowest concentration of an assay that can be measured with an interassay CV of 20%.

We applied the concept of functional sensitivity to the IMx Tacrolimus II assay. We first determined the analytical detection limit with 10 tacrolimus-free whole blood samples and calculated as the mean plus 2 standard deviations. Similar to reports by others (1)(2), the analytical detection limit was 0.8 µg/L. We then added tacrolimus to five whole blood samples and also used clinical specimens of four patients under tacrolimus immunosuppression. The mean tacrolimus concentrations of these samples varied between 2.3 and 7.5 µg/L. To obtain an interassay CV, frozen aliquots were run each day for a maximum of 10 days. As in routine laboratory testing, the samples were run singly and in duplicate. The CVs were correlated with the mean tacrolimus concentration and fitted with a polynomial equation (second order, Microcal^TM Origin^TM, Ver. 4.1). A functional sensitivity of 3.1 µg/L for single measurements and 1.9 µg/L for duplicate measurements, respectively, was calculated.

Considering the functional sensitivity, subtherapeutic tacrolimus concentrations in the long-term course can be detected by the new IMx Tacrolimus II assay with an acceptable imprecision. In the long-term course of liver-grafted patients, however, tacrolimus concentrations should always be measured in duplicate.

References

1. Wallemacq PE, Leal T, Besse T, Squifflet J-P, Reding R, Otte J-B, et al. IMx Tacrolimus II vs IMx Tacrolimus microparticle enzyme immunoassay evaluated in renal and hepatic transplant patients. Clin Chem 1997;43:1989-1991.[Free Full Text]
2. Cannon RD, Wong SHY, Hariharan S, Adams MB, Johnson CP, Roza AM, et al. Clinical evaluation of an improved sensitivity tacrolimus (FK 506) assay by microparticle enzyme immunoassay [Abstract]. Clin Chem 1997;43:S213.
3. Michel MC, Heemann U, Philipp T. Comparison of old and new IMx assays for monitoring of tacrolimus levels. Transpl Int 1997;10:409-410.[ISI][Medline] [Order article via Infotrieve]
4. Spencer CA. Thyroid profiling for the 1990s: free T₄ estimate or sensitive TSH measurement. J Clin Immunoassay 1989;12:82-89.