Plasma adrenocorticotropic hormone (ACTH) concentrations in ponies measured by two different assays suggests seasonal cross-reactivity or interference

BackgroundAnalysis of plasma adrenocorticotropic hormone concentration [ACTH] aids diagnosis of pituitary pars intermedia dysfunction (PPID). Comparisons of the validated chemiluminescent-immunoassay (CI) and immunofluorescent (IF) assays are limited. ObjectivesTo compare the results of [ACTH] analysis by CI and IF methods of samples collected in autumn and spring and assess cross-reactivity. Study designMethod comparison. MethodsPlasma from nonlaminitic ponies was analysed concurrently using the IF and CI methods in autumn and the following spring. Diagnostic thresholds for the IF method were derived using ROC curves and Youden indices to correspond with CI thresholds. Assay specificity was assessed using commercially available ACTH fragments and degradation products of endogenous ACTH. ResultsCI and IF methods yielded different results (P<0.001); mean differences (CI-IF), (95% confidence intervals): Autumn (n=99) 38.6 (30.6-46.5)pg/ml, Spring (n=88) 5.1 (3.9-6.3)pg/ml. The association between CI and IF results differed in autumn and spring, consistent with seasonally dependent cross-reactivity or interference. Good (=0.66-0.74) agreement was obtained for binary interpretation in spring between IF and CI using thresholds of >24 and >29pg/ml respectively and in autumn between IF and CI using thresholds of >27 and >47pg/ml respectively or >33 and >77pg/ml respectively. Of 88 ponies with both spring and autumn samples, 56 (64%) exceeded a published autumn CI threshold (>47pg/ml), of which 39 (70%) were below the equivalent threshold (<29pg/ml) the following spring without treatment. The CI assay showed apparent increases in [ACTH] following addition of CLIP (ACTH 18-39). Degradation of ACTH during storage affected the assays differently. Main limitationsLimited numbers of PPID cases were included. Immunoreactivity of commercially available peptides may differ from their endogenous equivalents. ConclusionsThe methods yielded different absolute values but the agreement for binary classification was good. An altered pituitary secretome in autumn that affects apparent [ACTH] values is likely. The Summary is available in Spanish - seeSupporting information.