Hormone stability in human whole blood
Introduction
Preparation of plasma samples for hormone analysis is not always possible immediately after venesection. The practice in an ideal setting in a specialized endocrine test center is usually the immediate centrifugation of blood specimens at 4°C. In reality, however, many specimens are collected in hospital wards or in external surgeries where a delay before plasma separation from red blood cells occurs frequently. As a referral laboratory specializing in the measurement of human hormones, we have no control over the collection procedures and integrity of the majority of incoming specimens. We are often asked about the suitability of specimens that have had delayed processing for the measurement of particular hormones. While it may be ideal to recommend a re-bleed, this may be inconvenient to the patient. It may even be impossible and have implications for diagnosis and patient management if, for example, a baseline value is required but treatment or surgery has occurred. Information on hormone stability in whole blood has been reported for some of the analytes that we measure [1], [2], [3], [4] although using dissimilar methods of analysis. This study was performed to provide information, relevant to our current hormone testing methods, on restrictions of time and temperature before separation of whole human blood on the subsequent levels of 17 hormones that are measured routinely in our clinical laboratory. We used EDTA plasma as this is the preferred matrix for hormone testing in our laboratory and that in which our reference range values were derived. We also found improved stability, particularly at higher temperatures, for several hormones in EDTA plasma compared to serum aliquots [5]. The end points studied included our recommended practice of separation within 30 min, a 6 h period that represents the time it may take for blood to reach a separation facility from a ward or surgery and a 24 h period that allows for an overnight delay in blood separation. The temperatures were chosen to reflect blood held under refrigeration or in ice/water and samples left at room temperature in a controlled environment such as a laboratory or hospital ward (temperature ranges 22–24°C).
Section snippets
Collection of samples
Blood samples from nonfasting “normal” healthy volunteers (80 ml per volunteer, 8–11 volunteers per analyte) were collected into tubes (chilled or at room temperature) containing ethylenediaminetetra-acetic acid (EDTA) at the Outpatient Bleeding service, Department of Endocrinology, Christchurch Hospital. The study was approved by The Canterbury Ethics Committee, Christchurch, New Zealand. Two 10 ml baseline samples were centrifuged immediately at 4°C. The remaining 10 ml blood tubes were held
Results
Four of the hormones tested, ACTH, insulin, AVP and VIP, showed a significant regression of mean concentration with respect to the time delay before blood separation. All remaining analytes were not significantly affected. Table 1 lists the number of volunteers per test, the range of values studied, the significance and the direction of the mean rate of change in hormone concentration. The calculated times for the median concentration of relevant hormones to change to either 90% or 110% of
Discussion
In this study we investigated the effects that the time and temperature at which whole human blood is held before separation of the plasma fraction has on the subsequent concentration of 17 hormones. Our definition of a 10% change in concentration as being significant is arbitrary and was chosen to exceed assay variability and as unlikely to influence clinical diagnosis. Most of the hormones investigated showed no significant alteration in plasma concentration when EDTA whole blood was left
Acknowledgements
The authors thank staff of the Endocrine Test Center, Christchurch Hospital, for collecting the blood samples, Caroline Smales for co-ordinating the treatment phase of the study, the technical staff of Endolab, Christchurch Hospital, for performing the hormone assays and Dr TG Yandle for validation of the aldosterone assay.
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