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Technical Briefs |
1
St. George’s Hospital Medical School, London SW17 0RE, United Kingdom
2
St. Bartholomew’s & The Royal London School of Medicine & Dentistry, London EC1M 6BQ, United Kingdom
3
The Queen Elizabeth Hospital, Woodville, South Australia 5011, Australia
4
Georg-August-Universität, D-37075 Göttingen, Germany
5
University of Texas Health Center, Houston, TX 77030
6
University of Pennsylvania Medical Center,
Philadelphia PA 19104
a address correspondence to this author at: Analytical Unit, St. George’s Hospital Medical School, London SW17 0RE, UK
Of late there has been a re-evaluation of therapeutic drug monitoring (TDM) strategies for optimizing cyclosporine (CsA) dosing in organ transplant recipients. Following the widespread introduction of the microemulsion formulation of CsA (Neoral®; Novartis Pharma), there has been a renewed interest in approaches to TDM that are based on the original observations of Lindholm and Kahan (1). These authors demonstrated that total exposure to CsA, as reflected by the area under the concentration-time curve (AUC), was a better predictor of outcomes than predose (trough) CsA concentrations. Furthermore, several studies have shown that the AUC can be estimated with good reliability by means of a limited sampling strategy (2)(3)(4).
Recently, clinical studies utilizing CsA measurements made at single or multiple time points in the early period (0–6 h) after CsA ingestion have shown the potential of such measurements for improving clinical outcomes compared with the traditional, predose, approach (5)(6)(7). These studies have made recommendations for target CsA concentration ranges at either specific postdose time points (2 or 3 h) or for limited AUC measurements in the period 0–6 h post dose. The recommendations were based on particular immunoassay methods and were for either kidney or liver transplant patients. We would like to raise two issues that may require further investigation before these new target CsA concentrations are adopted by other centers.
The first issue relates to the choice of analytical method used to
measure CsA. Currently, there are six analytical techniques in common
use for the measurement of CsA, and several more are in development
(8)(9). These analytical methods differ in
their accuracy and specificity for the measurement of the parent CsA
molecule in any one sample, and the average difference between two
methods can be as much as 57%. This between-method difference itself
is not constant and can be much larger, depending on such factors as
transplant type, the time after transplantation, and liver function.
Fig. 1
shows the mean values for the ratio between measurements of CsA
made with immunoassays with a high specificity for the parent compound
and HPLC. Although there is a trend for each immunoassay
method to give a higher value relative to HPLC, this relative
difference is not constant for two different pooled samples from kidney
transplant patients; both pooled samples were prepared from samples
that had been collected as predose (trough) samples. The variability
between methods is less well documented for samples collected in the
early period after CsA dosing. Thus, the application of target CsA
concentration ranges based on a published study that used a
radioimmunoassay to measure CsA in samples collected at, e.g., 2 h
post dose, in a center that utilizes a fluorescence polarization
immunoassay would need to be validated to document whether these
between-method differences are larger or smaller at such time
points.
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The second issue relates to our concern about the possible lack of adherence to best-practice analytical guidelines for handling samples containing high CsA concentrations. A recent proficiency testing survey of 125 clinical laboratories that were challenged with a blood sample containing a high concentration of CsA (added drug concentration, 2000 µg/L) produced a broad range of values (1082–3862 µg/L) although the laboratories had been alerted to the approximate concentration of CsA in the sample before analysis (10). This large variability was noted in a sample to which CsA had been added; the variability could be even greater in patient samples because CsA metabolites may not dilute in a linear fashion in immunoassays. In our opinion, the large variability in the analysis of high concentrations of CsA is, in part, attributable to a lack of on-site validated dilution guidelines. Blood samples containing high CsA concentrations, particularly those collected ~2 h after dosing, often require dilution before analysis because the CsA concentration in these samples may be higher than that of the highest CsA calibrator supplied with the assay. This is illustrated by the experience of two of us (R.G.M. and L.M.S.) for the measurement of CsA in samples collected 2 h post dose. For kidney transplant patients (R.G.M.), the median CsA concentration was 761 µg/L, (range, 143-2300 µg/L; n = 56) with the Emit® immunoassay (Dade Behring); for heart transplant patients (L.M.S.), the median CsA concentration was 1303 µg/L (range, 720-2211 µg/L; n = 35) with a validated HPLC assay. It is worth noting that the highest calibrator supplied with the Emit assay is only 500 µg/L. Thus, if the dilution step is not carried out using a validated procedure that has been shown to be linear across a wide range of concentrations, the resulting CsA concentration may be inaccurate. In turn, when applied to a TDM approach that is designed to estimate the absorbance profile, these values may introduce unacceptable inaccuracies into the estimate.
These issues need to be addressed further. The differences between the results produced by the various immunoassay methods, which produce a variable bias compared with a selective technique, require careful scrutiny at sample time points other than the traditional predose (trough) measurement. As shown above, and from interlaboratory comparisons (11), there is ample evidence demonstrating differences between analytical techniques for trough CsA concentration measurements, but these differences need to be studied for samples collected in the period 1–6 h after ingestion of CsA. We will be investigating this issue more fully in a series of controlled studies to be performed in the near future.
In addition, we hope that the manufacturers of commercially available CsA analytical systems will respond to changes in CsA TDM practices. For this, they should address the need for an increase in the ranges of their assay calibrators, as well as ensuring that validated dilution protocols are available for their customers.
Footnotes
References
The following articles in journals at HighWire Press have cited this article:
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  R. G Morris Immunosuppressant Drug Monitoring: Is the Laboratory Meeting Clinical Expectations? Ann. Pharmacother., January 1, 2005; 39(1): 119 - 127. [Abstract] [Full Text] [PDF]
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 J. M. Juenke, P. I. Brown, F. M. Urry, and G. A. McMillin Specimen Dilution for C2 Monitoring with the Abbott TDxFLx Cyclosporine Monoclonal Whole Blood Assay Clin. Chem., August 1, 2004; 50(8): 1430 - 1433. [Full Text] [PDF]
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F. Streit, V. W. Armstrong, and M. Oellerich Rapid Liquid Chromatography-Tandem Mass Spectrometry Routine Method for Simultaneous Determination of Sirolimus, Everolimus, Tacrolimus, and Cyclosporin A in Whole Blood Clin. Chem., June 1, 2002; 48(6): 955 - 958. [Full Text] [PDF]
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B. G. Keevil, D. P. Tierney, D. P. Cooper, and M. R. Morris Rapid Liquid Chromatography-Tandem Mass Spectrometry Method for Routine Analysis of Cyclosporin A Over an Extended Concentration Range Clin. Chem., January 1, 2002; 48(1): 69 - 76. [Abstract] [Full Text] [PDF]
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 C. Kimchi-Sarfaty, J. Kasir, S. V. Ambudkar, and H. Rahamimoff Transport Activity and Surface Expression of the Na+-Ca2+ Exchanger NCX1 Are Inhibited by the Immunosuppressive Agent Cyclosporin A and by the Nonimmunosuppressive Agent PSC833 J. Biol. Chem., January 18, 2002; 277(4): 2505 - 2510. [Abstract] [Full Text] [PDF]
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