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Cyclosporin Measurement – who cares about inaccuracy?

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Page 1: Cyclosporin Measurement – who cares about inaccuracy?

Received: 10 October 2000Accepted: 6 April 2001

Presented at “Antwerp 2000: Quality in the Spotlight in Medical Laboratories” Conference, 19–20 October 2000, Antwerp, Belgium

Abstract Most medical laboratoriesmeasure the immunosuppressivedrug cyclosporin using one of anumber of commercial immunoas-says, or high-performance liquidchromatography (HPLC). The cali-bration of these assays is based onmaterial supplied by the kit manu-facturers or prepared in-house. Wehave examined inaccuracy for themeasurement of cyclosporin in sam-ples spiked to known concentrationsand the impact of any inaccuracy onthe results for cyclosporin measure-ment in pooled samples from pa-tients prescribed the drug. The datawere from the International Cyclo-sporin Proficiency Testing Scheme,based on aliquots of cyclosporin-freeblood to which known amounts ofthe drug had been added or aliquotsof pooled samples collected from pa-tients receiving cyclosporin. Com-

pared with the results using HPLC,the immunoassays had a median biaswhich ranged from –4.5% to 8.2%for the spiked samples. When pooledsamples from patients were analysedthe percentage difference from themeasured HPLC value, allowing forassay inaccuracy, was as high as29.9%. It is concluded that inaccura-cy is a factor in between-assay per-formance for this measurement andthat proficiency testing schemesshould attempt to put more emphasison this aspect of assay performance.

Keywords Cyclosporin · Proficiency testing · Calibration andreference methods

Accred Qual Assur (2001) 6:405–408© Springer-Verlag 2001 GENERAL PAPER

David W. HoltKirsty DennyTerry LeeAtholl Johnston

Cyclosporin Measurement – who cares aboutinaccuracy?

Introduction

There is broad agreement amongst clinicians that doseadjustment of the immunosuppressive agent cyclosporinshould be based, at least in part, on blood concentrationmeasurements of the drug [1, 2]. A number of commer-cial immunoassays based on antibodies with varyingspecificity for the parent compound are available, to-gether with assays based on high-performance liquidchromatography (HPLC) with either UV or mass-spec-trometric detection [3, 4]. It is generally assumed thatHPLC assays for cyclosporin are the “gold standard”,since they are less likely to be biased by interferencewith cyclosporin metabolites than are the immunoassays

[5]. The calibration of these assays is based on materialsupplied by the kit manufacturers or prepared in-house.Previously we have noted that these calibrators may varyin comparative performance [6]. Thus, inaccuracy is anissue, which may be clinically important when dealingwith a critical-dose drug such as cyclosporin [7]. Thispaper examines inaccuracy for the measurement of cy-closporin, using data for the measurement of cyclosporinin samples spiked to a known concentration with thedrug. We then looked at the potential for inaccuracy toinfluence the results for the measurement in pooledblood samples from patients receiving cyclosporin.

D.W. Holt (✉ )Director, Analytical Unit, St George’s Hospital Medical School, London SW17 0RE, UKe-mail: [email protected]: +44-20-8767 9687

K. DennySt George’s Hospital Medical School, London, UK

T. LeeSt George’s Hospital Medical School, London, UK

A. JohnstonSt Bartholomew’s and The Royal LondonSchool of Medicine and Dentistry, London, UK

Page 2: Cyclosporin Measurement – who cares about inaccuracy?

Methods

Data from the International Cyclosporin ProficiencyTesting Scheme for the 25 month period June 1998 toJune 2000 were examined [8]. The scheme supplies threeblinded samples of blood each month. Typically, two arehuman whole blood spiked with cyclosporin to a nomi-nal concentration and the third is a pooled sample ofwhole blood from patients receiving the drug followingorgan transplantation. The data are analysed with respectto the analytical technique used. Data for this study werebased on the following samples:

One aliquot of cyclosporin-free blood.45 aliquots from cyclosporin-free blood to which knownamounts of the drug had been added. The nominal cyclo-sporin concentrations of the samples (number of aliquotsif >1) were 50, 100 (5), 150 (5), 200 (3), 250 (4), 300(6), 350 (2), 400 (5), 450 (2), 500 (4), 600 (4), 650, 700(2) and 800µg/l.29 aliquots from pools of blood collected from patientswho were receiving cyclosporin. These comprised 20 poolsfrom heart, 3 from liver and 6 from kidney transplant pa-tients.

Five assays with a high specificity for cyclosporin wereused during the study period. These were: high-perfor-mance liquid chromatography (HPLC), the CYCLO-Trac(tm) SP (DiaSorin) radio-immunoassay (RIA), theAxSYM(tm) (Abbott Laboratories) fluorescence polar-ization immunoassay (FPIA), the TDx(tm) (Abbott Lab-oratories) FPIA and the homogeneous enzyme multipliedimmunoassay (EMIT) from Dade Behring Diagnostics.

Statistical analysis of the data was performed as fol-lows:

Spiked samples – the measured value vs. nominal valueand percentage difference from spiked value vs. nominalvalue were calculated for each method. Data are dis-played as Box and Whisker plots.Pooled patient samples – the measured value vs. themeasured HPLC value and the percentage differencefrom the measured HPLC value were calculated for eachmethod. Again, data are displayed as Box and Whiskerplots.

Results

Across the 25-month period, on average, 398 centres permonth returned results. The percentage of centres usingeach method in both June 1998 and June 2000 are shownin Fig. 1. There was an increase in the proportion of lab-oratories using the AxSYM(tm) assay from 5% in June1998 to 18% in June 2000. Apart from a small number ofblunders, most centres reported a result of <10µg/l for

the cyclosporin-free blood sample. Spiking accuracy isillustrated by a graph of the results for the weighed-invalue, against the measured value obtained by HPLC,Fig. 2.

The results for the spiked samples showed that theHPLC and EMIT assays gave no significant differencefrom the weighed in values; the mean difference forHPLC was 0.5% (95%CI: –0.8 to +1.7%) and for EMITit was 1% (95%CI: –0.2 to +2.1%). The RIA assay had asmall positive inaccuracy of 2.7% (95%CI: +1.1 to+4.3%), and the AxSYM(tm) assay had a larger, nega-tive, inaccuracy of –4.5% (95%CI: –5.4 to –3.3%). Theleast accurate method was the TDx(tm) assay, with alarge positive inaccuracy of 8.2% (95%CI: +7.2 to+9.3%). The results are displayed as a Box and Whiskerplot in Fig. 3. The results for the pooled samples frompatients are illustrated in Fig. 4, which shows the per-centage difference from the measured HPLC value, byanalytical method. The EMIT and RIA show a similarpositive bias relative to HPLC; the mean difference for

406

Fig. 1 Cyclosporin analytical methods used by participants in theInternational Cyclosporin Proficiency Testing Scheme in June1998 and June 2000. EMIT – homogeneous enzyme multipliedimmunoassay from Dade Behring Diagnostics, HPLC – high-per-formance liquid chromatography, RIA – CYCLO-Trac(tm) SP(DiaSorin) radio-immunoassay, AxSYM – (Abbott Laboratories)fluorescence polarization immunoassay, TDx (Abbott Laborato-ries) FPIA

Fig. 2 Mean HPLC cyclosporin measurement versus nominalconcentration added to 45 blank blood samples

Page 3: Cyclosporin Measurement – who cares about inaccuracy?

Fig. 4 Cyclosporin assay method differences from HPLC resultfor pooled patient samples. The Box and Whisker plot shows thepercentage difference from the HPLC result for the measurementof cyclosporin in blood aliquots from 29 pooled samples from pa-tients receiving the drug

we were not employing a reference method [10], so wefelt the best way to check the accuracy of our materialwas to compare the mean values generated by centres us-ing HPLC against the weighed-in value. The results sug-gested that our spiked samples were very close to the ex-pected values. The inaccuracy of the centres using HPLCor EMIT was negligible for spiked samples, but therewas a small positive inaccuracy for the results fromthose using the RIA. Both the FPIA assays had larger in-accuracies – AxSYM(tm), –4.5%, and TDx(tm), +8.2%.It is worth noting that the calibrators for the FPIA areproduced by the same manufacturer but did not agreewhen compared with HPLC. A recent publication specu-lated that the values for the calibrators supplied with theAxSYM assay may be biased by a correction factor de-signed to improve the apparent specificity of this assay[11]. It was in the measurement of patient samples, con-taining both cyclosporin and its metabolites, that the big-gest differences were seen. Compared with measure-ments made by HPLC, the EMIT results were, on aver-age, 8.6% higher and the RIA 10.5% higher. The differ-ence between EMIT and RIA were consistent with theirrelative performance for spiked samples. However, bothFPIA assays showed a larger divergence, not only fromHPLC but also from each other. The AxSYM(tm) assayhad a 16.4% positive bias against the HPLC value forpatient samples, and the TDx(tm) 38.1%. Allowing forassay inaccuracy, these figures correspond to 21% forAxSYM(tm) and 29.9% for TDx(tm).

Conclusions

These data underline the need for certified reference ma-terial from an independent source and the use of validat-ed reference methods which can be used to assign valuesto proficiency testing material. Reference methodsshould be based on a chromatographic technique linkedto mass-spectrometric detection. It is possible that thecommon practice whereby proficiency testing schemesanalyse data with respect to method means could behelping to perpetuate method differences, since kit man-ufacturers are then able to focus on precision, rather thaninaccuracy, when promoting their product [12]. It is clearthat inaccuracy is a factor in between-assay performancefor this measurement and that proficiency testingschemes should attempt to highlight the problem by cir-culating both spiked samples and patient-derived materi-al. It is also possible that a similar problem is associatedwith the other immunosuppressant drugs which are mea-sured as a guide to therapy [13].

407

Fig. 3 Cyclosporin accuracy by assay method. The Box andWhisker plot shows the percentage inaccuracy for the measure-ment of cyclosporin in 45 blank blood samples to which knownamounts of the cyclosporin had been added

EMIT was 8.6% (95%CI: +6.9 to +10.4%) and for RIAit was 10.5% (95%CI: +8.1 to +12.9%). The Ax-SYM(tm) assay had a larger bias of 16.4% (95%CI:+13.7 to +19.1%), and the TDx(tm) assay had the largestbias relative to HPLC of 38.1% (95%CI: +35.2 to+41.1%).

Discussion

Substantial differences in calibration for cyclosporin as-says were noted. These differences could introduce biasinto the clinical interpretation of cyclosporin measure-ments and, generally, require the introduction of methodspecific target concentration ranges [5]. They could alsomake comparison of between-centre data from majorclinical studies conducted on multiple sites difficult [9].This study does, of course, rely on the accuracy of theproficiency testing samples prepared by us. At the time,

Page 4: Cyclosporin Measurement – who cares about inaccuracy?

References

1. Sketris I, Yatscoff R, Keown P, Canafax DM, First MR, Holt DW et al.(1995) Clin Biochem 28:195–211

2. Kahan BD, Shaw LM, Holt DW, Grevel J, Johnston A (1990) ClinChem 36:1510–1516

3. Steimer W (1999) Clin Chem 45:371–381

4. Schutz E, Svinarov D, Shipkova M,Niedmann PD, Armstrong V, WielandE et al. (1998) Clin Chem 44:2158–2164

5. Oellerich M, Armstrong VW, KahanBW, Shaw L, Holt DW, Yatscoff R et al. (1995) Ther Drug Monit17:642–654

6. Johnston A, Holt DW. (1993) ClinChem 39:2532–2533

7. Johnston A, Keown PA, Holt DW.(1997) Ther Drug Monit 19:375–381

8. International Cyclosporin ProficiencyTesting Scheme: http://www.asil.de-mon.co.uk/ (accessed 28 December,2000).

9. Holt DW, Johnston A (1999) AccredQual Assur 4:427–30

10. Büttner J (1991) Eur J Clin Chem ClinBiochem 29: 223–225

11. Morris RG (2000) Ther Drug Monit22:160–162

12. Hurst AD (1998) Ann Clin Biochem35:12–18

13. Johnston A, Holt DW (1999) Br J ClinPharmacol 47:339–350

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