The Effect of Temperature and Aprotinin on the preanalytical stability of adrenocorticotrophic hormone

Abstract

Objective: The susceptibility of plasma adrenocorticotropic hormone (ACTH) to enzymatic degradation has limited the stability of ACTH. Many non-routine tests require the collection of blood on special proteinase inhibitor cocktails to preserve the integrity of the sample. This may provide benefits in sample collection and routine clinical practice. This study examined if a proteinase inhibitor, aprotinin, mixed with blood at the time of collection, would change the stability of ACTH in plasma at various temperatures for several hours and days using immunoassay. Methods: Sequential human blood samples were collected from 22 patients in EDTA collection tubes and EDTA plus aprotinin tubes, respectively. Plasma obtained was stored for 4 days at 4°C and on the other hand a second aliquot at room temperature. ACTH was measured immediately and after storage times of 1, 2, 4, 24 h, and 96 h at 4°C and other aliquots at room temperature. Results: Aprotinin plus K3EDTA-plasma samples centrifuged and stored at 4°C showed the best stability of ACTH up to 96 h compared to the ACTH concentration collected into K3EDTA tubes. Also, aprotinin plasma stored at room temperature showed no significant change according to baseline values. ACTH in plasma samples without aprotinin centrifuged and stored at 4°C declined in 2 h. Conclusion: The addition of aprotinin showed an improvement of the stability at 4°C/RT. Blood samples for ACTH measurement could be taken into blood tubes containing K3EDTA + aprotinin and without the need of keeping the sample on ice. These data indicate that aprotinin has a profound preservative effect upon plasma ACTH level and that it may be possible to submit unfrozen samples collected with this inhibitor to appropriate referral laboratories for analysis of ACTH immunoassay. From a practical standpoint, referral laboratories may wish to prepare collection tubes containing both EDTA and aprotinin for distribution to other laboratories. Key Words: ACTH, aprotinin, EDTA, stability, room temperature, preanalytical factors Conflict of Interest: The authors have no conflict of interest. ÖZETAmaç: Adrenokortikotropik hormonun (ACTH), enzimatik yıkıma uğramasındaki duyarlılık, ACTH' ın stabilitesini sınırlandırır. Birçok rutin olmayan testler, örneklerin bütünlüğünü korumak adına kanların özel proteaz inhibitör kokteyllerinde toplanmasını gerektirir. Bu da örnek-lerin toplanma ve rutin klinik alanda yararlar sağlayabilir. Bu çalışmada kan alma tüplerine eklenen bir proteinaz inhibitörü olan aprotininin, farklı sıcaklık derecelerinde ve farklı saatlerde, immunassay yöntemiyle ölçülen ACTH' in stabilitesinde değişiklik yapıp yapmadığı incelendi. Metod: Ardışık olarak 22 hastadan sırasıyla EDTA' lı ve EDTA+aprotinin' li örnek tüplerine kan alındı. Elde edilen plazmanın bir kısmı, 4°C' de ve kalan kısmı ise oda sıcaklığında dört gün boyunca depolandı. ACTH, hızla ve 4°C' de depolandıktan sonraki 1., 2., 4., 24. ve 96. saatlerde ve oda sıcaklığındaki diğer kısımlarda ölçüldü. Bulgular: Dört dercede da santrifüj e...

Feline plasma adrenocorticotropic hormone: validation of a chemiluminescent assay and concentrations in cats with hypercortisolism, primary hypoadrenocorticism and other diseases

“…Previous investigations in humans 30,31 and dogs 23,29 have suggested that the use of aprotinin, a protease inhibitor, may help prevent ACTH proteolytic degradation after sample collection. In our study, plasma ACTH values in the presence and absence of aprotinin were not significantly different in the three sets of samples analysed.…”

“…28 In clinical practice, however, this procedure may present logistical problems because samples for plasma ACTH measurement are frequently shipped to an external laboratory. Therefore, an additional aim of the present study was to determine if aprotinin, a protease inhibitor previously investigated for use in human and canine samples, 23,29–31 may help prevent ACTH degradation in feline plasma samples.…”