Study on the reproducibility of ACTH concentrations in plasma of horses with and without Pituitary Pars Intermedia Dysfunction (PPID)

The evaluation of plasma ACTH and the dexamethasone suppression test are considered the methods of choice to evaluate the course of therapy of pituitary pars intermedia dysfunction (PPID). Sampling protocols as well as vacutainers for analysis differ between the laboratories, To evaluate the reproducability of plasma ACTH measurement between four different laboratories (A, B, C, D) in Germany as well as within the laboratories themselves, ten horses with previously diagnosed PPID and four healthy horses were sampled and analyzed. Each laboratory received two differently labeled samples of each horse which had been drawn at the same time (blinded samples). Sampling was performed in the morning at the same time. The sampling vacutainers (with and without addition of coagulation and proteinase inhibitors) and postage of the samples was performed according to laboratory standards. In one laboratory the influence of the time of centrifugation (immediately after taking blood versus after one hour) was determined. The samples were processed and analyzed according to laboratory protocols. Determination of ACTH levels was performed using chemiluminescence immunoassay. In total 132 blood samples were analyzed. The results of doubled blood samples of the same horse showed a standard deviation ranging from +/- 6 to +/- 27 pg/ml within the laboratories (empty set 19,29 pg/ml). The standard deviation of the repeatability of. the variation coefficient was 13,48%. Blood samples of the same horse resulted in ACTH levels of 121 pg/ml in the first probe and in <5 pg/ml in the second probe. Standard deviation of measured ACTH values between the laboratories was +/- 26,4 pg/ml (empty set 27,44 pg/ml).The standard deviation of the reproducibility of the variation coefficient was 18,36%. In a 20 year old gelding the lowest ACTH value was 60.9 pg/ml whereas the highest measured value was 108 pg/ml. Immediate centrifugation of blood samples resulted in significantly higher ACTH values at an average of 11.6 pg/ml. The additional use of proteinase inhibitors (aprotinine) showed no influence on ACTH levels in this study.