Cyclosporin whole blood immunoassays (AxSYM, CEDIA, and emit): a critical overview of performance characteristics and comparison with HPLC.
The CYP3A-dependent metabolism of CsA produces a variety of known metabolites. Whether these metabolites are still toxic or whether they possess substantial immunosuppressive efficacy is still controversial (9,10). On the other hand, these metabolites are of substantial importance with regard to TDM of CsA because all immunoassays currently used for whole blood CsA determination suffer from cross-reactivity with these metabolites to varying degrees (11,12). Several consensus meetings have established recommendations for TDM of CsA (13,14) during the last decade. At the most recent meeting, the Lake Louise Consensus Conference on Cyclosporin Monitoring, an overview from 35 transplant centers around the world showed that 26% used a specific HPLC method for CsA determination, whereas most other centers used relatively specific methods for CsA measurement based on radioimmunoassays (14%) or nonisotopic immunoassays (57%) (14). Because the reproducibility and analytical specificity for the parent drug is critical for an acceptable CsA immunoassay, the following performance criteria have been recommended in the consensus documents (13,14): (a) imprecision [less than or equal to]10% at 50 [micro]g/L and <5% at 300 [micro]g/L; and (b) for accuracy, comparison with the reference method (a validated HPLC procedure) should yield a slope of 0.9-1.1, an intercept of -15 to 15 [micro]g/L, and [S.sub.y/x] [less than or equal to]15 [micro]g/L, calculated with bivariate, preferably nonparametric procedures (e.g., Passing-Bablok method) (15). Two newly specific CsA whole blood assays have become available in 1997 and 1998. The monoclonal antibody-based fluorescence polarization immunoassay (mFPIA) CsA assay has been adapted for the AxSym immunoanalyzer (Abbott). A homogeneous enzyme immunoassay for CsA quantification in whole blood, which does not require sample extraction, has been developed using CEDIA[TM] technology (Boehringer Mannheim). The latter is not yet FDA-approved. We have evaluated these assays in terms of analytical performance characteristics and compared them to a validated HPLC method with ultraviolet detection (16). In addition, the Emit Assay (bade Behring Diagnostica), which has been FDA-approved since 1997, was also included in the investigation.
Materials and Methods
Measurements using the mFPIA on the AxSYM (Abbott) were carried out on an analyzer that also serves for routine measurements of other drugs in our laboratory. EDTA-anticoagulated whole blood, calibrators, or control material were pretreated according to the manufacturers' instructions, and the measurement was then carried out automatically by the AxSYM. Calibration was based on six calibration points (in duplicate) and was carried out whenever the deviation of the controls was >10% of the assigned value. This was not necessary during the 4-week study period.
CEDIA is based on the spontaneous association of a short recombinant NHZ terminal [beta]-galactosidase fragment (apeptide) and a recombinant [beta]-galactosidase monomer with a deletion near the [NH.sub.2] terminus to form active enzyme tetramer. CsA is chemically attached to the a-peptide, and an anti-CsA antibody binds to both CsA in the sample and CsA coupled to the a-peptide. Because the latter process interferes with the formation of active enzyme, the amount of CsA in the sample is directly proportional to the residual enzyme activity. The pretreatment step for the CEDIA is different to that of the other immunoassays, because a separation step is not necessary. After lysis of the whole blood, CEDIA (Boehringer) was carried out on a Hitachi 917 automated analyzer in batch analyses, without concomitant measurements of other analyses. The calibration was based on two calibration points (in duplicate), and the system was recalibrated whenever the deviation of the controls was >10% of the assigned value or whenever reagent bottles had to be changed as recommended by the manufacturer. Mean calibration frequency was once a week during the study period.
The Emit procedure (bade Behring) was preceded by a methanol extraction according to the manufacturer's protocol. The closed secondary cup was directly placed on the rack of one of two dedicated Cobas Mira Plus analyzers (Roche). The calibration was based on five calibration points (in duplicate) and was done whenever the deviation of the controls was >10% of the assigned value. The mean frequency of calibration is 2 weeks in our laboratory.
The HPLC procedure was carried out as described elsewhere (16). Briefly, 1 mL of whole blood, calibrators, or controls was mixed with internal standard solution. After hemolysis, liquid-liquid extraction, and solid phase extraction, HPLC analyses were carried out with a Supelcosil LC1 (50 X 4.5 mm, 5 [micro]m, Supelco) column, heated to 72 [degrees]C. Calibration was done within each analysis, using in-house calibrators with precisely weighed concentrations of CsA and cyclosporin D as internal standard. Peak height from ultraviolet detection at 214 nm was used for the construction of the calibration curve. Measurements were performed on a Gynkotek HPLC system with a diode array detector (Gynkotek). HPLC measurements were accepted if the ultraviolet spectra of the CsA and cyclosporin D peaks showed an identity with standard CsA and cyclosporin D spectra of >98%. The detection limit, defined as signal-to-noise ratio of 5:1, was 25 [micro]g/L. The within-run imprecision (CV) of this HPLC method was <5.8%, whereas between-day imprecision was <7.8% at CsA concentrations between 100 and 500 [micro]g/L.
The manufacturers' controls, pooled sample material, and independent commercially available control materials (Lyphocheck Whole Blood Control, Bio-Rad) were used for the evaluation of precision, both within-run and between-day, and for reproducibility studies. Purified metabolites (AM1, AM9, and AM4N; Novartis GmbH) were used for estimation of cross-reactivity. Influences of the different pretreatment procedures were excluded by using drug-free whole blood specimens with CsA added as well as using the metabolites in methanol solutions.
For recovery experiments, a CsA standard preparation (US Pharmacopeia) was added to a human whole blood pool to give concentrations of 50, 100, and 400 [micro]g/L.
For method comparisons of CsA, we used 100 samples each from kidney, heart, and liver recipients. If the deviation of the CsA concentrations determined with the AxSYM, CEDIA, and Emit was >15%, samples were rerun on each systems to exclude random errors. Only reproducible results were accepted for statistical calculations. Values of CsA trough concentrations above the dilution limit of an assay were excluded from calculations.
The detection limits of the immunoassays were defined and estimated by the method according to Kaiser (17) as the concentration that is above zero, with an error probability <2.5% (The concentration that equals the mean plus 3 standard deviations of the signal produced by analyte-free samples).
The nonparametric regression procedure developed by Passing and Bablok (15) was used for the estimation of the structural relationship between the immunoassays and the HPLC method in clinical samples (EVAPAK, Ver. 2.08, Boehringer). The 95% confidence interval for the estimates of slope and intercept is given, together with the 95% median distance of the residuals. In addition, the standard error of the residuals ([S.sub.y|x]) of the standard principal component model is also presented for comparison with the recommendations of the consensus documents (13,14).
The scatter of method comparison data was visualized according to the recommendations of Bland and Altman (18). The differences between the methods are plotted against the mean of the methods.
Analytical sensitivity was assessed using the method of critical difference (19). The critical difference is defined as 2 X [square root of (2)] X SD (as assessed by imprecision profiles) for CsA measurement with the particular method and represents the minimal difference between two measurements that a method is able to discriminate.
The detection limits according to Kaiser (17) were 13 [micro]g/L for the AxSYM, 25 [micro]g/L for the CEDIA, and 17.0 [micro]g/L for the Emit procedure.
IMPRECISION AND ACCURACY
Imprecision within series and analytical specificity. Samples of pooled whole blood were measured in a series of 20 within one analytical run (Table 1). A low CsA concentration was added to one pool; an identical higher (290 [micro]g/L) CsA concentration was added to the other three (pools 2-4). Two of these latter whole blood pools were also supplemented with AM1 (pool 3, 1000 [micro]g/L) and AM9 (poo14, 500 [micro]g/L).
The calculated average cross-reactivity from the data in Table 2 for the AxSym was 8.6% for AM1,14.5% for AM9, and not detectable for AM4N. The CEDIA showed a cross-reactivity of 6.3% for AM1, 27% for AM9, and 5% for AM4N. The Emit assay had no detectable cross-reactivity with AM1 and AM4N; however, it had ~8% crossreactivity with AM9.
Imprecision between runs. Imprecision data are given in Table 2. The CsA procedures on the AxSYM as well as the Emit assay fulfill the recommended consensus criteria of imprecision (12,13). In contrast, the imprecision of the CEDIA determined in a control sample with ~90 [micro]g/L CsA exceeded the limit of 10% that was recommended for concentrations at the low end of the therapeutic range.
Cross-reactivity with metabolites. Cross-reactivity towards the metabolites AM1, AM9, and AM4N is shown in Table 3. Purified metabolites were added to drug-free whole blood without CsA in the samples. All assays displayed a cross-reactivity with AM9; the highest cross-reactivity was seen with the CEDIA and the lowest with the Emit. In the case of AM1, the AxSYM assay showed the highest cross-reactivity, followed by CEDIA, whereas the Emit did not cross-react with AM1 at concentrations up to 1 mg/L. Only the CEDIA procedure was found to have a detectable cross-reactivity towards AM4N. The Emit assay showed the least cross-reactivity with all metabolites tested. The use of the new nonvolatile pretreatment reagent for the Emit (Cyclosporine Sample Pretreatment Reagent, Dade Behring), which will serve as a substitute for methanol beginning in 1998 (anticipated launch date for United States is November 1998, Food and Drug Administration filing in progress), did not alter the cross-reactivity toward the metabolites (data not shown). The cross-reactivity to AM1 and AM9 derived from CsA-free whole blood pools with metabolites added (Table 3) are in close agreement to those values in the presence of ~290 [micro]g/L CsA (pools 3 and 4, Table 1), The differences in the calculated cross-reactivities with and without CsA present did not exceed the limits of the analytical sensitivity.
Dilution linearity. Dilution linearity of the immunoassays was investigated with the appropriate dilution protocol of each assay, using either the highest calibrator or a patient sample. For dilutions up to 1:8, the linearity was acceptable with all of the immunoassays (deviation from target value <10%).
Recovery in human blood. To assess the recovery of the CsA whole blood assays, a human whole blood pool was prepared and supplemented with a precisely weighed CsA standard preparation (US Pharmacopeia). The HPLC method used for comparison in patients' samples displayed the best recovery (mean, 100.2%), followed by the Emit assay (mean, 98.0%), which was independent of the target CsA concentration. In contrast, the percentage of recovery of both AxSYM (mean, 96.6%) and particularly CEDIA (mean, 93.7%) showed a lower recovery at the lower CsA concentrations. The details of the recovery experiments are given in Table 4.
COMPARISON OF METHODS
Kidney recipients. The comparability of the CsA whole blood concentration measured with the immunoassays vs results from HPLC in samples from kidney recipients are visualized in Fig. 1, using the Bland-Altman difference plot.
The structural relationship (estimates from the Passing-Bablok model with 95% confidence intervals in parentheses) between the HPLC method and the immunoassays as well the median distances of residuals (MD95), [S.sub.y|x], and mean differences (d%) are given in Table 5. It is obvious that Emit and CEDIA show a very similar overestimation of ~20%; however, the Emit has a much narrower dispersion of residuals. Of the immunoassays evaluated, AxSYM displayed the highest overestimation compared with HPLC.
Heart recipients. The method comparison results with samples derived from heart recipients are very similar to those from kidney recipients. The scatters of the differences between HPLC and the immunoassays are shown in Fig. 2, and the statistical data are given in Table 5.
Similar to the results for kidney recipients, all methods overestimate CsA in whole blood from heart recipients. The results from the CEDIA are comparable with those from the Emit; however, the latter displays the narrower dispersion of residuals. Again the AxSYM assay showed the highest discrepancy to HPLC.
Liver recipients. The most challenging samples for CsA assays are those from liver recipients because these samples are the most likely to contain high concentrations of CsA metabolites. The results are presented in Fig. 3 and Table 5. It is obvious that the difference from HPLC is indeed the highest of all patients groups. Of the three immunoassays, the Emit procedure displayed the lowest overestimation compared with HPLC and the smallest [S.sub.y|x] (dispersion of residuals). Comparable values were seen for the AxSYM and the CEDIA, which both showed a substantially higher overestimation compared with HPLC than the Emit.
TDM for CsA is strongly recommended for the individualization of dosage in CsA-based immunosuppression. Because the turnaround time should allow results to be reported within one dosing interval, which is usually 12 h, most large transplant centers prefer semiautomated immunoassays for CsA monitoring (14). All methods, however, require sample pretreatment, compromising the opportunity of automation. In the case of the AxSYM CsA whole blood assay, two pretreatment solutions must be pipetted, as opposed to only one with the Emit. The precision of the AxSYM method is, however, comparable with that of the Emit assay. The very easy sample pretreatment with the CEDIA, which does not require a separation step, makes it easy to perform and reduces the technical time. However, this test has a between-day imprecision at CsA concentrations <100 [micro]g/L that exceeds the recommendations of the consensus documents (13,14).
[FIGURE 2 OMITTED]
In view of our knowledge that in certain circumstances (e.g., cholestasis in liver recipients) large amounts of CsA metabolites may accumulate (20), analytical specificity towards the parent drug is very critical for effective CsA monitoring with immunoassays. Two immunoassays are thought to fulfill the specificity criteria recommended in the consensus documents (13,14), the 3H-radioimmunoassay originally produced by Sandoz and the Emit that is commercially available from Dade Behring (11). According to the data from the present evaluation in a larger number of samples, this interpretation should be regarded with caution. None of the commercial nonisotopic immunoassays available today totally fulfills the consensus recommendations for accuracy and specificity of CsA measurement.
Dilution linearity seems to be a less critical issue for CsA monitoring, because the measuring range of all assays spans the therapeutic ranges for CsA trough concentrations (Ax5YM,13-800 [micro]g/L; CEDIA, 25-620 [micro]g/L; Emit, 17-500 [micro]g/L). On the other hand, it has recently been discussed that with the new microemulsion formulation of CsA (Neoral) that a reduction in acute rejection rate after kidney transplantation may be achieved if CsA concentrations measured 2 h after drug intake are maintained between 800 and 1200 [micro]g/L (8). Because such 2-h samples will usually exceed the upper limit of the assays, dilution will be unavoidable. The linearity is acceptable with all immunoassays for dilutions up to 1:8.
[FIGURE 3 OMITTED]
Cross-reactivity toward CsA metabolites AM1 and AM4N remains higher with the new AxSYM assay than with the CEDIA, whereas the latter assay shows the highest cross-reactivity towards AM9. The low recovery for the US Pharmacopeia cyclosporin standard in human whole blood in the CEDIA assay at lower CsA concentrations may partially mask this lack of specificity. The lowest cross-reactivities and the best recoveries were found with the Emit. This is especially obvious in samples from liver recipients, where a 47% mean deviation from the HPLC method was observed with the AxSym and 43% with the CEDIA, as opposed to 31% deviation with the Emit. In kidney and heart recipients, both the Emit and the CEDIA displayed a similar mean deviation from HPLC of ~22%, whereas the results from the AxSym were on average ~34% higher than HPLC results in this patients. The new AxSYM CsA assay has an advantage in that this immunoanalyzer is in widespread use, which makes the test readily available. In addition, it is easy to perform and is highly reproducible. This assay is, therefore, an attractive alternative for laboratories already using the established mFPIA running on the TDx/FLx system.
The CEDIA assay has a very easy sample preparation that requires less technical time and can be performed, in principle, on several clinical chemistry analyzer models. The comparatively poor precision of the test even in batch analyses seems to be a critical issue, and it can be speculated that running the test in a random access mode may even lead to deterioration. The Emit test has a sample preparation that requires separation but uses only one reagent, which is available in each laboratory. If the new nonvolatile pretreatment reagent is used, this test can also be adapted to clinical chemistry analyzers and so would require a technical time between that needed for the other immunoassays. Concerning the performance criteria, the Emit has the best specificity towards the parent drug, and it shows the lowest overestimation of the investigated immunoassays compared with the HPLC method. The between-day imprecision also fulfills the consensus criteria (13,14).
Taken together, the data clearly show that, currently, the performance of nonisotopic immunoassays for CsA monitoring is still not satisfactory. In particular, analytical specificity has to be substantially improved. In liver recipients with unexpectedly high trough concentrations because of the accumulation of CsA metabolites, a validated HPLC assay is still the method of choice and should be available in centers dealing with such samples. From an economic point of view, the additional costs for confirmatory HPLC analyses in such cases must be calculated against the follow-up costs arising from incorrect therapeutic decisions guided by erroneous CsA monitoring. In the majority of the other clinical situations, the use of an immunoassay with the performance characteristics of the Emit seems to be a practical compromise.
We thank Jutta Engelmayer, Regina Martin, and Martha Onufrejow for skillful technical assistance. Special thanks go to Kurt Wonigeit, Medizinische Hochschule Hannover, Germany, for providing us with specimens from liver graft recipients.
Received February 12, 1998; revision accepted June 25, 1998.
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EKKEHARD SCHUTZ,  DOBRIN SVINAROV,  MARIA SHIPKOVA,  PAUL-DIETER NIEDMANN,  VICTOR W. ARMSTRONG,  EBERHARD WIELAND,  and MICHAEL OELLERICH 
 Abteilung Klinische Chemie, Georg-August-Universitat Gattingen, D-37075 Gattingen, Germany.
 Department of Clinical Laboratory, Medical University of Sofia, 1341, Bulgaria.
* Address correspondence to this author at: Abteilung Klinische Chemie, Zentrum Innere Medizin, Georg-August-Universitat, Robert Koch Strasse 40, D-37075 Gattingen, Germany. Fax 49-551-398551; e-mail email@example.com.
 Nonstandard abbreviations: CsA, cyclosporin A; TDM, therapeutic drug monitoring; and mFPIA, fluorescence polarization immunoassay (monoclonal antibody).
Table 1. Within-run imprecision (a) and specificity of CsA whole blood assays. Pool1 Pool2 Pool3 Pool4 AxSym Mean, [micro]g/L 60.5 271 341 334 CV, % 8.2 7.2 6.6 5.3 CEDIA Mean, [micro]g/L 65.1 304 342 429 CV, % 8.2 7.8 4.2 4.3 Emit Mean, [micro]g/L 62.1 291 281 321 CV, % 9.7 3.1 5.5 4.9 HPLC Mean, [micro]g/L 63.1 296 293.5 286 CV, % 6.2 6.4 4.2 6.8 (a) Within-run imprecision of the whole blood CsA assays determined with pooled whole blood, with CsA added. Pools 3 and 4 are identical to pool whereas 1000 rig/L AM1 was added to pool 3 and 500 [micro]g/L AM9 was added t pool 4. Table 2. Between-day imprecision and accuracy of CsA whole blood assays. AxSym (a) Control 1 Control 2 Target, [micro]g/L 70 300 Mean, [micro]g/L 70.8 300 CV, % 5.8 1.7 CEDIA Control 3 Control 4 Target, [micro]g/L 71.5 197 Mean, [micro]g/L 72.4 189 CV, % 11.0 5.5 Emit Control 3 Control 4 Target, [micro]g/L 88 199 Mean, [micro]g/L 88 196 CV, % 6.5 4.8 HPLC Control 5 Control 6 Target, [micro]g/L 97.5 445 Mean,[micro]g/L 96.0 441 CV, % 6.8 7.6 (a) For AxSYM, the control material of the manufacturer Emit measurements were performed with third party control material (Lypho-check Whole Blood Control, Bio-Rad). Method-specific target values are displayed. HPLC control material was purchased from Merck Merck/Recipe), with assigned HPLC target values. Calculations were from 15 control determinations on consecutive working days. Table 3. Cross-reactivity of AxSym and Emit CsA whole blood assays with purified metabolites. Metabolite, [micro]g/L AM9 (a) AM1 (a) AM4N (a) AxSym 1000 136 83 <12.5 500 82 46 <12.5 CEDIA 1000 252 61 48 500 151 34 32 Emit 1000 81 <17 <17 500 40 <17 <17 (a) Apparent CsA concentration (rig/L) measured in drug-free whole blood with purifed CsA metabolites added. Means of two independent series. Table 4. Recovery of CsA standard preparation added to human whole blood. (a) Target HPLC Emit AxSYM CEDIA 50 I [micro]g/L Mean, [micro]g/L 50.1 48.9 46.4 43.3 SD 1.85 2.77 5.28 5.48 Recovery, % 100.2 97.8 92.8 86.6 100 [micro]g/L Mean, [micro]g/L 100.9 96.9 96.3 91.0 SD 3.03 6.79 5.78 5.38 Recovery, % 100.9 96.9 96.3 91.0 400 [micro]g/L Mean, [micro]g/L 397.6 396.1 406.6 415.5 SD 17.1 10.81 23.67 17.54 Recovery, % 99.4 99.0 101.7 103.9 (a) Means from 10 determinations are given for each pool and method. Table 5. CsA concentrations measured with immunoassays (y-values) compared with HPLC (x-values). n Slope (a) Intercept (a) Kidney AxSYM 99 1.17 (1.04-1.32) 13.2 (-4.4-26.9) CEDIA 99 1.19 (1.00-1.39) -3.5 (-22.1-18.0) Emit 99 1.07 (0.97-1.19) 16.2 (0.90-28.5) Heart AxSYM 97 1.09 (0.95-1.24) 30.0 (11.5-50.0) CEDIA 97 1.01 (0.88-1.16) 21.9 (1.0-40.0) Emit 97 1.01 (0.90-1.14) 22.0 (5.4-38.8) Liver AxSYM 95 1.30 (1.18-1.42) 11.0 (0.4-24.3) CEDIA 95 1.28 (1.17-1.41) 10.2 (-3.6-24.6) Emit 95 1.15 (1.03-1.26) 14.7 (3.1-25.8) MD95 (b) [S.sub.x|y] (c) Mean d% (d) Kidney AxSYM 37.1 19.2 32.0 CEDIA 48.1 24.0 22.5 Emit 32.9 16.1 23.9 Heart AxSYM 53.2 23.8 33.9 CEDIA 42.0 22.6 20.5 Emit 40.4 19.5 20.2 Liver AxSYM 40.2 22.6 47.5 CEDIA 45.1 22.9 42.9 Emit 41.4 19.4 31.2 (a) Values in parentheses after slope and intercept represent 95% confidence limits. (b) MD95, 95~ median distance of the residuals of the Passing-Bablok regression. (c)[S.sub.y|x], SD of the residuals of the principle component method. (d) Mean d%, mean difference of the measurement of either method to HPLC results in percentage of HPLC value.
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|Title Annotation:||Drug Monitoring and Toxicology|
|Author:||Schutz, Ekkehard; Svinarov, Dobrin; Shipkova, Maria; Niedmann, Paul- Dieter; Armstrong, Victor W.; W|
|Date:||Oct 1, 1998|
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