Short communication: A note on the correction for the effect of freezing on the outcome of pregnancy-associated glycoprotein measurement in blood and serum of cows

Abstract

Early pregnancy detection is a measure of considerable economic relevance for dairy cattle breeders, and analysis of pregnancy-associated glycoprotein (PAG) values in blood is one of the methods implemented in practice. Starting from d 30 postconception, cows are considered to be pregnant at PAG levels of 2.0 ng of PAG/mL of blood and higher. However, little is known about preanalytic sources of errors that might affect PAG values. Based on blood samples from 65 dairy cows, the present study showed that freezing of samples, such as may be the case during shipping in wintertime, will lower PAG values considerably. Therefore, a Bland-Altman analysis was used to derive a correction factor. Overall, the mean differences (± standard deviation) between frozen and respective fresh samples was -5.5 ± 7.4 ng of PAG/mL of blood and 0.9 ± 6.1 ng of PAG/mL of serum. However, the Bland-Altman plot revealed a concentration-dependent effect of freezing on PAG values with higher variability and larger declines at higher PAG levels. Therefore, to minimize chances of false-negative results, different correction factors are suggested for different levels of PAG (e.g., based on the upper bound of the 95% confidence interval 0.67 for PAG levels between 2.0 and 3.9 ng of PAG/mL and 0.25 for PAG levels between 4.0 and 7.9 ng of PAG/mL). With these concentration-dependent correction factors, implementation into practice will be possible. The accuracy is adequate because no quantitative information but qualitative results (pregnant vs. nonpregnant) are required. However, due to larger chances of false-negative results, the application of the correction factor should only be a last resort if temperature exposure of a sample is unknown.

Short communication: The effect of storage conditions and storage duration on milk ELISA results for pregnancy diagnosis

“…Previous studies have investigated the effect of freezing on PAG ELISA results for blood and serum (Stahmann et al, 2013). The results of these studies showed that PAG levels in blood and serum decreased following freezing and storage for 2 wk.…”

“…Bland-Altman plots are used to measure agreement between 2 tests and we acknowledge that using this approach to compare results before and after storage is not the exact application of this statistical measure. Stahmann et al (2013) examined the effect of freezing serum and blood samples for 2 wk on PAG level as measured by ELISA. They noted a decline in PAG levels that was concentration dependent, with higher variability and greater declines at higher initial PAG levels.…”

Pre-Analytic Sources of Error with the Serum PAG-Pregnancy Test for Cows

“…The discrepancy with the findings of Experiment 2 might be ascribed to differences in PAG molecules prevailing at an early vs. an advanced stage of pregnancy (Green et al, 2005), pertaining, according to Klisch et al (2005), to differences in molecular mass or arrangement of carbohydrate side chains. As reported in Stahmann, Gauly, Holtz and Koenig-von Borstel (2013), this phenomenon necessitates application of different correction factors to compensate for the distorting effect of freezing on PAG measurements at different PAG levels.…”