Validation study of canine serum cortisol measurement with the Immulite 2000 Xpi cortisol immunoassay

Abstract

We report the results of validation of canine serum cortisol determination with the Immulite 2000 Xpi cortisol immunoassay (Siemens), with characterization of precision (CV), accuracy (spiking-recovery [SR] bias), and observed total error (TEo = bias + 2CV) across the reportable range, specifically at the most common interpretation thresholds for dynamic testing. Imprecision increased at increasing rate with decreasing serum cortisol concentration and bias was low, resulting in increasing TEo with decreasing serum cortisol concentration. Inter-laboratory comparison study allowed for determination of range-based bias (RB) and average bias (AB). At 38.6 and 552 nmol/L (1.4 and 20 μg/dL), between-run CV was 10% and 7.5%, respectively, and TEo ~30% and ~20%, respectively (TEo remained similar regardless of the considered bias: SR, RB, or AB). These analytical performance parameters should be considered in the interpretation of results and for future expert consensus discussions to determine recommendations for allowable total error (TEa). Importantly, the commonly used thresholds for interpretation of results were determined ~40 y ago with different methods of measurements and computation, hence updating is desirable. Quality control material (QCM) had between-run imprecision of 4% for QCM1 and 7% for QCM2; the bias was minimal for both levels. Acceptable QC rules are heavily dependent on the desired TEa for the QCM system (TEa_QCM), itself limited by the desired clinical TEa. At low TEa_QCM (20-33%), almost no rules were acceptable, whereas at high TEa_QCM (50%), almost all rules were acceptable; further investigation is needed to determine which TEa_QCM can be guaranteed by simple QC rules.

Keywords: Cushing; accuracy; bias; cortisol; dogs; endocrinology; precision; total error.

Figure 1.

Canine serum cortisol validation study overview: a spiking-recovery phase, a between-laboratory comparison phase, and a quality control material (QCM) phase. LDD = low-dose dexamethasone suppression test; MSU-VDL = Veterinary Diagnostic Laboratory of Michigan State University; TEo = total observable error; TVMDL = Texas A&M Veterinary Medical Diagnostic Laboratory.

Figure 2.

Reportable range study, with simple linear regression for within-run spiking-recovery canine serum cortisol. The simple linear regression yields a slope of 0.953 (close to 1), an intercept of 0.538 (close to 0). The coefficient of determination R² of 0.997 (close to 1) shows that the assessed range is sufficient. Linearity was confirmed between 0.25 and 50 μg/dL.

Figure 3.

A. Within-run precision = f [serum cortisol]. Evolution of within-run (n = 4) CV (%) across the serum cortisol concentration. Spiked cortisol levels for the linearity study were run in quadruplicate, allowing assessment of the CV evolution across the concentration: CV remained low (1.5–7%) from 1,380 nmol/L (50 μg/dL) to 69 nmol/L (2.5 μg/dL), before increasing at an increasing rate at lower concentrations. B. Serum cortisol spiking-recovery bias = f [serum cortisol]. Evolution of the spiking-recovery bias (%) across the serum cortisol concentration. Spiked cortisol levels for the linearity study were run in quadruplicate, and the mean was used to calculate the recovery and the bias percentages. The spiking-recovery bias remained minimal (–7% to +4%) across the reportable range. C. Serum cortisol TEo_SR = f [serum cortisol]. Evolution of TEo (%) across the serum cortisol concentration. Within-run CV (from Fig. 3A) and spiking-recovery bias (from Fig. 3B) were combined to calculate TEo across the serum cortisol concentration.

Figure 4.

Inter-laboratory comparison study for canine serum cortisol (n = 20), with Passing–Bablok regression (solid line) and its 95% CI (dotted lines). MSU-VDL = Veterinary Diagnostic Laboratory of Michigan State University; TVMDL = Texas A&M Veterinary Medical Diagnostic Laboratory.

Figure 5.

Bland–Altman comparison for the inter-laboratory canine serum cortisol comparison study (n = 21), with simple linear regression of the differences between laboratories. Both laboratories (TVMDL and MSU-VDL) used the same method of measurement: Immulite 2000 Xpi. There was one freezing cycle added before measurement by the MSU-VDL. The simple linear regression of the difference followed the equation: y = −0.0319x + 0.0236 (dotted line). Thus, there was nearly no constant bias; the proportional bias was negligible. Indeed, the latter was minimal at low concentration, and it would not impact clinical interpretation at higher concentration. MSU-VDL = Veterinary Diagnostic Laboratory of Michigan State University; TVMDL = Texas A&M Veterinary Medical Diagnostic Laboratory.

Figure 6.

Example of utilization of a normalized operational process specifications (OPSpec) chart, at N = 2 levels of QCM, and P_ed of the system = 90%, for QC rule validation of L4 (38.6 nmol/L [1.4 μg/dL]) in green, and QCM1 (193 nmol/L [7.0 μg/dL]) in purple. In this example, we are considering the averaged bias (from the comparison study) for QC rule determination of L4 used as a QCM (L4_AB). First, CV and bias are determined, and TEo is calculated. Then, a given level of TEa is chosen. Here, when TEo was <20%, TEa was chosen as 20% (QCM1); when TEo was >20%, TEa was chosen as 33% (L4_AB). Then, CV and bias are expressed as % of TEa (“normalized”), and those values are plotted on the x-axis and y-axis of the OPSpec chart, respectively, as the “operating point.” All the QC rules to the right of the operating point are candidates for use with the specified P_ed and P_fr; all the QC rules left from the operating point are not. Thus, there is no candidate QC rule for L4_AB. For QCM1, 1_2.5s or 1_3s/2_2s/R_4s would be candidate QC rules; 1_2s should be avoided because of the P_fr of 0.09 (see discussion). AB = averaged bias; CV = coefficient of variation; L4 = level 4 in this study, corresponding with 1.4 μg/dL; N = number of levels tested for QC; OPSpec = operational process specifications; P_ed = probability of error detection; P_fr = probability of false rejection; QCM = quality control material; R = number of repeats for each QC level; TEa = allowable total error; TEo = observed total error.