Brief Communication  Communication brève Comparison of commercial progesterone assays for evaluation of luteal status in dairy cows André Broes, Stephen J. LeBlanc Abstract — Serum progesterone (P4) concentration was measured using 2 methods, and ovaries were examined by ultrasonography (US) to determine the presence of a corpus luteum (CL) in 101 lactating dairy cows. The concordance correlation coefficient between the 2 methods (Ovucheck and Immulite P4 assays) was high (r c = 0.94); agreement between the assays on presence of a CL based on P4 . 1 ng/mL was excellent (Kappa = 0.88) and of each assay with US was good (Kappa = 0.63 for each). Résumé — Comparaison des épreuves commerciales de mesure de la progestérone pour l’évaluation du statut lutéinique chez les vaches laitières. La concentration de la progestérone sérique (P4) a été mesurée en utilisant deux méthodes et les ovaires ont été examinés par échographie pour déterminer la présence d’un corps jaune chez 101 vaches laitières en lactation. La concordance du coefficient de corrélation entre les deux méthodes (épreuves Ovucheck et Immulite P4) était élevée (rc = 0,94); la concordance entre les épreuves biologiques sur la présence d’un corps jaune basée sur P4 . 1 ng/mL était excellente (Kappa = 0,88) et celle de chacune des épreuves biologiques avec l’échographie était bonne (Kappa = 0,63 pour chacune). (Traduit par Isabelle Vallières) Can Vet J 2014;55:582–584


easurement of circulating progesterone (P4) concentration in dairy cattle is useful to determine the stage of the estrous cycle and may be applied to determine whether a cow is anovular, in estrus, or to guide reproductive management interventions. Circulating concentrations of P4 during an ovulation synchronization program influence the probability of pregnancy (1–3). The rate of increase of P4 concentration after insemination is predictive of pregnancy (3,4). Quantification of the concentration of P4 may be informative, but estimation of the presence or absence of a functional (secreting P4) corpus luteum (CL) is often sufficient to be useful for clinical decisions in veterinary practice, management of reproduction, or classification of cows in a research study. The presence of a functional CL is reflected by serum or plasma progesterone . 1 ng/mL (5), and this status is of practical relevance. The presence of functional CL may be assessed with 89% accuracy (relative to plasma P4 . 1 ng/mL) using detection of a CL . 22 mm in diameter with ultrasound imaging of the ovary 24 d after

Biovet, Saint-Hyacinthe, Quebec J2S 8W2 (Broes); Department of Population Medicine, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1 (LeBlanc). Address all correspondence to Dr. Stephen LeBlanc; e-mail: [email protected] Support for this study was provided by Biovet. Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. 582

insemination (6). Cows in estrus have serum P4 , 1 ng/mL; lower P4 concentration at insemination is associated with a higher probability of pregnancy (3). Conversely, among cows with a CL, higher P4 concentrations during diestrus in the days before luteolysis and in the week following insemination are associated with better follicle and embryo development, the latter through effects of P4 on endometrial gene expression and function (7). Several assay methods are available for P4 in blood or milk. Radio-immunoassay (RIA) has been widely used and considered the reference method (5), but RIA involves the use of radioactive materials and thus requires specialized facilities and equipment. Therefore, a variety of enzyme-linked immunosorbent assays (ELISA) and commercial kits have been developed and validated (5). These methods include chemiluminescent enzyme immuno­assay (CLEIA) and colorimetric ELISA read by absorbance spectrometers. The relatively simpler methods and equipment requirements of the latter may make them more applicable for veterinary practice. The objective of this study was to compare the performance of a commercial ELISA kit (Ovucheck Plasma; Biovet, St. Hyacinthe, Quebec) for measurement of serum P4 in dairy cows with a previously validated commercial reference test (Immulite, Siemens, Mississauga, Ontario), and with ultrasonography to classify the presence of a CL. The ELISA does not have published validation data in cattle. The study subjects were a convenience sample of 101 lactating Holstein cows from 1 large dairy farm in Ontario. All the data were collected on 1 day. There were 89 cows examined for pregnancy diagnosis at least 28 d following insemination, 35 of which were diagnosed pregnant and 54 were not pregnant. CVJ / VOL 55 / JUNE 2014


Figure 1.  Passing-Bablok correlation plot for progesterone concentrations from a chemiluminescent (CLEIA) reference assay on the x-axis and an ELISA assay on the y-axis. There were 101 samples (1 per cow), shown with the linear regression line (dashed central grey line), regression 95% confidence limits (dotted grey lines), data 95% prediction limits (outer dashed lines) and identity line (x = y; solid black line).

Figure 2.  Bland-Altman plot (n = 101 samples; 1 per cow), with the mean progesterone concentration as measured by a chemiluminescent (CLEIA) reference assay and an ELISA assay on the x-axis, plotted against the difference in progesterone concentrations determined by the 2 methods on the y-axis. The mean difference and the 95% confidence limits around the difference are indicated by the horizontal lines.

An additional 12 cows between 3 and 10 days postpartum were examined as “negative controls” expected to not have a CL and to have low P4. The ovaries of each cow were examined by an experienced veterinarian using transrectal ultrasonography to detect the presence of a CL, defined as a distinct greyish echogenic structure in the ovarian stroma, approximately circular in appearance, with or without a central anechoic cavity, and estimated to be . 22 mm in diameter. At the same time, a blood sample was collected from the coccygeal vessels into an evacuated sterile tube without anticoagulant (Vacutainer; Becton Dickinson, Franklin Lakes, New Jersey, USA). The tubes were left at room temperature for about 30 min to allow blood coagulation and then placed on ice packs in a cooler. Samples were centrifuged at 1400 3 g for 10 min at 20°C to collect serum within 5 h of blood collection and were frozen for 3 wk at 220°C until analyzed. Samples were shipped frozen to Biovet, stored at 220°C, then thawed in a refrigerator, vortexed, and analyzed by both assays on the same day. Progesterone concentration was determined by ELISA (Ovucheck Plasma) and CLEIA (Immulite). The ELISA is a monoclonal antibody kit labeled for use with plasma or serum and uses standards with P4 concentrations of 0 (blank), 1, 2.5, 5, and 10 ng/mL; the log P4 concentration has a linear relationship with the optical density of standards, read at 405 nm in a microplate absorbance reader. The CLEIA is a polyclonal antibody kit using calibration standards at 0.2 and 20 ng/mL. The intra-assay and inter-assay coefficients of variation (CV) were 10.5% and 9.6% for the ELISA and 5.5% and 7.6% for the CLEIA. This CLEIA has been validated in bovine samples against the standard RIA method (8) and was taken as the reference test against which the ELISA was compared; RIA was not available at the lab where the ELISA and CLEIA were performed, but the correlation of this CLEIA with RIA in cattle was previously reported to be 0.95 (8). Cross-reactivity with other steroids is reported in the product inserts to be , 1% for tested compounds with the exceptions of deoxy-corticosterone

(CLEIA: 1.2%; ELISA: 3%), hydroxy-progesterone (CLEIA: 17a form: 1.04%; ELISA: 11a form: 66%), and pregnandione (ELISA: 3 to 5%). No cows in the present study were receiving exogenous synthetic progesterone. Both assays were conducted by experienced technicians according to the instructions of the manufacturers. Each sample was measured once and technicians were blinded to the cows’ status. The data included cow number, presence of a CL as assessed by ultrasound (yes or no), pregnancy status, and the concentration of P4 from each of the ELISA and CLEIA. The data were analyzed using SAS, version 9.3 (SAS, Cary, North Carolina, USA). Linear regression was performed, Lin’s concordance correlation coefficient (9) was calculated and a Passing-Bablok plot was drawn (10), comparing the values from the ELISA to the CLEIA results. Using only correlation coefficients may not be suitable for evaluation of diagnostic test performance (11); therefore, a Bland-Altman plot was used to assess the residuals between the results from the ELISA and the CLEIA. Classification of the presence of a CL was based on P4 . 1 ng/mL for each assay. Agreement of the classification of presence of a CL between the 2 assays and between each assay and ultrasonography was assessed with contingency tables and the Kappa statistic for agreement beyond chance. Values of Kappa 0 to 0.20 indicate slight agreement, 0.21 to 0.40 fair, 0.41 to 0.60 moderate, 0.61 to 0.80 substantial, and 0.81 to 1 almost perfect agreement (9). The relationship of P4 concentrations between the ELISA and CLEIA is presented in Figure 1. The regression equation was: ELISA = 0.46 1 0.97 3 CLEIA. Correlation was high (Pearson’s r = 0.95 and the coefficient of determination r 2 = 0.90) but the intercept differed statistically from 0 (P = 0.004) and the slope from 1 (P , 0.001), indicating small differences between the P4 measurement methods. These statistical differences are not practically important; agreement between the tests as described below relate better to the clinical performance of the test. The concordance correlation coefficient (Lin’s rc) was 0.94 [95%

CVJ / VOL 55 / JUNE 2014



confidence interval (CI): 0.91 to 0.96]. At concentrations between 0.2 and 2 ng/mL, results from the ELISA were, on average, 0.3 6 0.4 ng/mL lower than from the CLEIA, while between 3 and 8 ng/mL values from the ELISA were, on average, 0.9 6 0.7 ng/mL higher than those from the CLEIA (Figure 1). A Bland-Altman plot of the magnitude and pattern of differences between the ELISA and CLEIA is presented in Figure 2. For the ELISA, 97% of the results fell within the 95% CI of the mean difference between the assays. Based on the CLEIA, 73 of 101 cows had P4 $ 1 ng/mL and 28 cows had P4 , 1 ng/mL. The ELISA data provided 68 samples in agreement with the CLEIA that P4 was $ 1 ng/mL, 28 in agreement that P4 was , 1 ng/mL, 5 in which CLEIA was $ 1 ng/mL but ELISA was lower, and no samples to the converse. Therefore, classification of P4 concentration $ 1 ng/mL resulted in almost perfect agreement between the two assays (Kappa = 0.88, 95% CI, 0.78 to 0.98). If the previously validated CLEIA is taken as the reference test for classification of P4 $ 1 ng/mL, the sensitivity (correct classification of cows with P4 $ 1 ng/mL) of the ELISA was 93% (95% CI, 85 to 97%) and the specificity (correct classification of cows with P4 , 1 ng/mL) was 100% (95% CI: 88% to 100%). Examination of the 5 discordant P4 classifications revealed that in each case, the ELISA underestimated P4 relative to the CLEIA (the paired results were 0.9 and 1.1, 0.4 and 1.2, 0.9 and 1.4, 0.7 and 1.4, and 0.9 and 1.8 ng/mL, respectively; all 5 cows were not pregnant but in the latter 3 cases, a CL was seen on ultrasound). Agreement between the ELISA and US for the presence of a CL was “substantial” (Kappa = 0.63, 95% CI: 0.46 to 0.79). Agreement was the same between CLEIA and US (Kappa = 0.63, 95% CI: 0.47 to 0.80). Given P4 . 1 ng/mL from the CLEIA as the reference test, the sensitivity of ultrasound under the present conditions was 91% (95% CI: 81% to 95%) and the specificity was 72% (54% to 85%). In turn, relative to ultrasound the sensitivity and specificity of the ELISA were 86% (76% to 92%) and 79% (62% to 90%), respectively. However, the ultrasound assessments were performed by 1 veterinarian in 1 herd on 1 day, and may not be suitable for generalization for ultrasound for all operators or in different contexts. In a large study, ultrasound generally yielded high sensitivity (89 to 98%) relative to a gold standard of plasma P4 . 1 ng/mL, but low specificity (46% to 51%) if CL detection was subjective or qualitative; specificity was improved to 89% when the CL was measured and was . 22 mm (7). In the present case, sen-


sitivity was comparably high (91%) but specificity (72%) was intermediate, reflecting estimation of the diameter of the CL (as may commonly be done in veterinary practice) rather than formal and more time-consuming on-screen measurement with electronic calipers. Due to the strong concordance correlation and excellent agreement between P4 concentrations obtained from serum by the ELISA and the CLEIA, this ELISA is valid for measurement of progesterone and determination of CL status in lactating dairy cattle in clinical practice and in research applications.

Acknowledgments The technical support of Martine Bertrand, Mélanie St-Hilaire, Isabelle Caya (Biovet), and Christine Gutscher (Guelph) is greatly appreciated. CVJ

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CVJ / VOL 55 / JUNE 2014

Comparison of commercial progesterone assays for evaluation of luteal status in dairy cows.

Comparaison des épreuves commerciales de mesure de la progestérone pour l’évaluation du statut lutéinique chez les vaches laitières. La concentration ...
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