Comparison of plasma separation techniques prior to freezing on equine immunoreactive adrenocorticotrophic hormone

Summary Background Pituitary pars intermedia dysfunction (PPID) is the most common endocrine disorder affecting older horses and is commonly diagnosed by measurement of basal plasma adrenocorticotrophic hormone (ACTH) concentration. Current guidelines advise that whole blood or gravity‐separated plasma samples must not be frozen prior to analysis. Objectives The aim of this study was to determine the effect of gravity separation and specifically whether gravity‐separation prior to freezing would result in increased ACTH concentrations when compared with samples centrifuged prior to freezing. Study design Split‐sample, repeated‐measures experimental design. Methods Blood samples from 22 horses were subjected to four different sample preparation methods: centrifuged fresh samples, centrifuged frozen samples, gravity‐separated fresh samples and gravity‐separated frozen samples. Friedman's test with Dunn's multiple comparisons was used to make comparisons between sample preparation groups. Results Sample separation method (centrifugation vs. gravity separation) had a significant effect on ACTH concentration ( p < 0.0001). Samples that underwent centrifugation demonstrated no significant change in ACTH concentration following freezing (median % change of 3.9%, p > 0.99). In contrast, both gravity‐separated fresh samples (median % change of 23.9%) and gravity‐separated frozen samples (median % change of 14.0%) demonstrated significantly increased ACTH concentrations when compared with the reference centrifuged fresh samples ( p < 0.0001). From a clinical perspective, two samples that would have been initially classified as non‐PPID based on basal ACTH levels in centrifuged fresh samples were subsequently classified as PPID positive following freezing of gravity‐separated samples, while a further seven samples were reclassified into the equivocal diagnostic zone. Main limitations Small sample size and small number of PPID‐positive samples included in the study. Conclusions These findings demonstrate that gravity‐separated samples submitted to laboratories for ACTH analysis should undergo centrifugation prior to testing and support current recommendations that gravity‐separated samples should not be frozen prior to analysis.