Stability of cytokines,chemokines and soluble activation markers in unprocessed blood stored under different conditions

BackgroundBiomarkers such as cytokines, chemokines, and soluble activation markers can be unstable when processing of blood is delayed. The stability of various biomarkers in serum and plasma was investigated when unprocessed blood samples were stored for up to 24 h at room and refrigerator temperature.MethodsBlood was collected from 16 healthy volunteers. Unprocessed serum, EDTA and heparinized blood was stored at room (20–25 °C) and refrigerator temperature (4–8 °C) for 0.5, 2, 4, 6, 8, and 24 h after collection before centrifugation and separation of serum and plasma. Samples were batch tested for various biomarkers using commercially available immunoassays. Statistically significant changes were determined using the generalized estimating equation.ResultsIFN-γ, sIL-2Rα, sTNF-RII and β₂-microglobulin were stable in unprocessed serum, EDTA and heparinized blood samples stored at either room or refrigerator temperature for up to 24 h. IL-6, TNF-α, MIP-1β and RANTES were unstable in heparinized blood at room temperature; TNF-α, and MIP-1β were unstable in unprocessed serum at room temperature; IL-12 was unstable in unprocessed serum at refrigerator temperature; and neopterin was unstable in unprocessed EDTA blood at room temperature. IL-1ra was stable only in unprocessed serum at room temperature.ConclusionAll the biomarkers studied, with the exception of IL-1ra, were stable in unprocessed EDTA blood stored at refrigerator temperature for 24 h. This indicates that blood for these biomarkers should be collected in EDTA and if delays in processing are anticipated the unseparated blood should be stored at refrigerator temperature until processing.     

Blood Substrate Collection and Handling Procedures under Pseudo-Field Conditions: Evaluation of Suitability for Inflammatory Biomarker Measurement

Routine incorporation of blood-based biomarker measurements in population studies has been hampered by challenges in obtaining samples suitable for biomarker assessment outside of laboratory settings. Here, we assessed the suitability of venous blood left unprocessed for 4, 24, or 48 hours post-collection at either room temperature or 4°C for quantification of two biomarkers, Interleukin-6 (IL-6) and C-reactive protein (CRP). Blood samples were collected in both K₂EDTA tubes and a dedicated plasma-preservation tube, P100. Dried blood spot (DBS) samples from the same subjects were also collected in order to compare delayed-processing plasma performance against a popular alternative collection method. We found that K₂EDTA mean plasma concentrations of both IL-6 and CRP were not significantly different from concentrations in plasma processed immediately; this was observed for tubes stored up to 48 hours pre-processing at either temperature. Concentrations of IL-6 measured in P100 tubes showed significant time-dependent increases when stored at room temperature; otherwise, levels of IL-6 and CRP were similar to those found in samples processed immediately. Levels of CRP in DBS were correlated with plasma CRP levels, even when pre-processed blood was stored for up to 48 hours. These data indicate that plasma is suitable for IL-6 and CRP estimation under data collection conditions that involve processing delays.     

Effect of holding time and temperature of bovine whole blood on concentration of progesterone, estradiol-17beta and estrone in plasma and serum samples

Bovine jugular venous blood was collected, with and without heparin, and aliquoted into 140 12-ml tubes. Four subsamples (two heparinized and two coagulated) were centrifuged immediately (time zero) and plasma or serum was aspirated and stored at -20 degrees C. One-half of the remaining subsamples were stored at 4 degrees C and the other one-half at 25 degrees C (room temperature). At 1-h intervals (0 to 24 h), 6-h intervals (24 to 72 h) and at 96 and 120 h, four subsamples (heparinized and coagulated at both 4 degrees C and 25 degrees C) were centrifuged, plasma or serum was aspirated and stored at -20 degrees C. Whole blood incubation for 1 h at 25 degrees C reduced mean plasma and serum progesterone (P(4)) concentration (P<0.05). Similarly, whole blood incubation at 4 degrees C for 2 and 3 h, respectively, reduced mean plasma and serum P(4) concentration (P<0.05). No difference was found in mean P(4) concentration between plasma and serum samples harvested from whole blood incubated at 4 degrees C or 25 degrees C. Concentration of estradiol-17beta (E(2)) and estrone (E(1)) fluctuated over time, irrespective of holding temperature. There was a blood type, heparinized or coagulated, by time interaction (P<0.01) for both E(2) and E(1) concentrations It was concluded that incubation time and temperature between collection and centrifugation of bovine blood samples influenced the assayable P(4) concentration in both plasma and serum. In contrast, incubation temperature had no effect on assayable E(2) and E(1) concentrations, but assayable E(2) and E(1) over time were differentially affected, depending on whether plasma or serum was assayed.     

Sample handling and stability of hepatocyte growth factor in blood samples

As regards clinical studies performed on hepatocyte growth factor (HGF) during recent years, we have aimed in the present study to investigate the eventual differences in sample handling of this cytokine that might influence the results of serum concentrations. Venous blood from patients with current infectious diseases and controls was used in different sub-studies. Compared with samples separated within one hour, no significant changes in serum HGF levels were observed when whole blood stayed 4, or 24h at 6 degrees C before or 6h in room temperature after separation but HGF levels were significantly higher (P<0.01) when whole blood was kept at room temperature 4 and 24h before separation. Serum HGF was stable up to 20 freeze-thaw cycles. The serum concentrations of HGF were significantly higher than levels in the plasma (19%; P<0.05). A significant increase in serum HGF levels (12%, P<0.05) was observed after shaking the whole blood sample to a visible haemolysis, although the HGF concentration in blood cells was around half of that in serum. HGF tolerated storage at -70 degrees C for at least 4 months. We conclude that standardized methods in sample handling are important in the study of HGF concentrations in blood samples.     

Changes in porcine,ovine, bovine and equine blood progesterone concentrations between collection and centrifugation

The aim of this study was to determine if different methods of handling porcine, ovine, bovine and equine blood between collection and centrifugation influence measurable progesterone levels. A 2 × 2 × 5 factorial experiment was conducted for each species with heparin (with or without), temperature of incubation (4 and 22°C) and time of incubation (0, 6, 12, 24 and 48 h) as the main effects. Following centrifugation, plasma and serum samples were stored at ?20°C until progesterone concentrations were determined by radioimmunoassay. Method of handling porcine and equine blood between collection and centrifugation did not affect the levels of progesterone. However, heparinized blood held at 4°C resulted in the most consistent levels of progesterone over time. Progesterone levels were fairly consistent across time in the ovine blood by all methods of handling except heparinized blood incubated at 22°C. By 24 h after collection, plasma progesterone concentrations decreased by 50% for the ovine blood incubated at 22°C with heparin. Decreases were detected by all the methods of handling the bovine blood between collection and centrifugation. The rate of decline, however, was considerably faster for blood held at 22°C than blood held at 4°C. At 12–48 h after collection, the concentrations of progesterone averaged only 5% of the time 0 sample for blood incubated at 22°C. In contrast, at least 30% of the progesterone values in the time 0 sample were detected between 12 and 48 h of incubation for the blood held at 4°C.     

Stability of targeted metabolite profiles of urine samples under different storage conditions

Introduction

Few studies have investigated the influence of storage conditions on urine samples and none of them used targeted mass spectrometry (MS).

Objectives

We investigated the stability of metabolite profiles in urine samples under different storage conditions using targeted metabolomics.

Methods

Pooled, fasting urine samples were collected and stored at ?80 °C (biobank standard), ?20 °C (freezer), 4 °C (fridge), ~9 °C (cool pack), and ~20 °C (room temperature) for 0, 2, 8 and 24 h. Metabolite concentrations were quantified with MS using the AbsoluteIDQ? p150 assay. We used the Welch-Satterthwaite-test to compare the concentrations of each metabolite. Mixed effects linear regression was used to assess the influence of the interaction of storage time and temperature.

Results

The concentrations of 63 investigated metabolites were stable at ?20 and 4 °C for up to 24 h when compared to samples immediately stored at ?80 °C. When stored at ~9 °C for 24 h, few amino acids (Arg, Val and Leu/Ile) significantly decreased by 40% in concentration (P < 7.9E?04); for an additional three metabolites (Ser, Met, Hexose H1) when stored at ~20 °C reduced up to 60% in concentrations. The concentrations of four more metabolites (Glu, Phe, Pro, and Thr) were found to be significantly influenced when considering the interaction between exposure time and temperature.

Conclusion

Our findings indicate that 78% of quantified metabolites were stable for all examined storage conditions. Particularly, some amino acid concentrations were sensitive to changes after prolonged storage at room temperature. Shipping or storing urine samples on cool packs or at room temperature for more than 8 h and multiple numbers of freeze and thaw cycles should be avoided.

     

The effect of storage on whole blood chemiluminescence measurement of equine neutrophils

The aim of this study was to determine the effect of duration and temperature of sample storage on whole blood chemiluminescence measurement results. Venous blood from 18 clinically healthy Polish half‐bred horses aged 4 to 11 years were used in the study. Luminol dependent chemiluminescence (CL) was used to measure neutrophil oxygen metabolism in whole blood. Blood samples were examined for spontaneous CL and stimulated by a surface receptor stimulus as well as extra‐receptor stimulus. The assay was performed in two parallel experimental sets with samples stored at 4 and 22 °C, respectively. Whole blood CL was estimated at 2, 6, 24, 48, 72, 96 and 120 h after collection. The study demonstrated that temperature and duration of sample storage are factors that determine the quality of CL measurements of whole blood in horses. The study concluded that samples should be stored at 4 °C and the assay should be performed as early as possible. It was also shown that the viability period of horse blood for CL assays is relatively long. Material stored at room temperature for 24 h and even up to 48 h at 4 °C did not show any significant decrease in spontaneous or stimulated chemiluminescence. Copyright © 2012 John Wiley & Sons, Ltd.     

A comparison between the concentration of prostaglandin F in human plasma and serum

The concentration of prostaglandin F (PGF) has been measured in serum and plasma samples prepared under different conditions from the antecubital vein blood of 4 non-pregnant and 7 pregnant women. Prostaglandin F concentrations were less than 41 pg/ml in 19 samples of serum or plasma prepared by centrifugation within 30 minutes of collection. When the blood was allowed to clot at room temperature for 24 hours, highly variable, but usually markedly increased concentrations of PGF (<30 - 3020 pg/ml) were found in the serum. Plasma obtained from blood which stood at 23°C for 24 hours contained undetectable amounts of PGF in 4 out of 6 samples and less than 75 pg/ml in the 2 remaining samples. Plasma and serum obtained from blood which stood at 4°C for 24 hours contained less than 45 pg PGF/ml. These results show that (i) incubation of blood at room temperature may markedly elevate concentrations of PGF in serum, (ii) plasma samples rather than serum should be used for measurements of PGF concentrations.     

The influence of some sample handling factors on progesterone and testosterone analysis in goats

This study validated the use of commercially available radioimmunoassay kits for measuring the circulating progesterone and testosterone levels of goats. Progesterone and testosterone levels were then assayed in plasma which was collected from 23 does and 8 bucks. Collections from each animal were divided into three sodium fluoride-potassium oxalate (F/OX), one heparin, and one EDTA tubes and also into a tube without anticoagulant. Plasma from an F/OX tube was separated immediately from the blood cells by centrifugation. Serum or plasma was also separated after storage for 24 hours with F/OX, heparin or EDTA anticoagulant at 22 degrees C or with F/OX at 5 degrees C. A significant decline in assayable progesterone occurred in samples stored at 22 degrees C with each anticoagulant used and in the serum sample. Samples stored at 5 degrees C for 24 hours with F/OX anticoagulant contained concentrations of progesterone which did not differ significantly from those in samples where plasma was removed immediately. Assayable testosterone did not change with the anticoagulant used or vary with the storage temperature when F/OX tubes were stored at 5 degrees C and 22 degrees C for 24 hours. Results indicate that sample storage does influence levels of measured progesterone but not testosterone in goats. Progesterone assay is best done on plasma which is immediately separated from blood cells or on samples which are stored at 5 degrees C.     

Freeze-dried plasma proteins are stable at room temperature for at least 1 year

Thirty human EDTA plasma samples from male and female subjects ranging in age from 24 to 74 years were collected on ice, processed ice cold and stored frozen at ?80 °C, in liquid nitrogen (LN2), or freeze dried and stored at room temperature in a desiccator (FDRT) or freeze dried and stored at ?20 °C for 1 year (FD-20). In a separate experiment, EDTA plasma samples were collected onto ice, processed ice cold and maintained on ice ± protease inhibitors versus incubated at room temperature for up to 96 h. Random and independent sampling by liquid chromatography and tandem mass spectrometry (LC–ESI–MS/MS), as correlated by the MASCOT, OMSSA, X!TANDEM and SEQUEST algorithms, showed that tryptic peptides from complement component 4B (C4B) were rapidly released in plasma at room temperature. Random sampling by LC–ESI–MS/MS showed that peptides from C4B were undetectable on ice, but peptides were cleaved from the mature C4B protein including NGFKSHALQLNNR within as little as 1 h at room temperature. The frequency and intensity of precursors within ± 3 m/z of the C4B peptide NGFKSHALQLNNR was confirmed by automated targeted analysis where the precursors from MS/MS spectra that correlated to the target sequence were analyzed in SQL/R. The C4B preproprotein was processed at the N terminus to release the mature chain that was cleaved on the carboxyl side of the isoprene C2 domain within a polar C terminal sequence of the mature C4B protein, to reveal the thioester reaction site, consistent with LC–ESI–MS/MS and Western blot. Random sampling showed that proteolytic peptides from complement component C4B were rarely observed with long term storage at ? 80 °C in a freezer or in liquid nitrogen (LN2), freeze drying with storage at ? 20 °C (FD-20 °C) or freeze drying and storage at room temperature (FDRT). Plasma samples maintained at room temperature (RT) showed at least 10-fold to 100-fold greater frequency of peptide correlation to C4B and measured peptide intensity compared to samples on ice for up to 72 h or stored at ? 80 °C, LN2, FDRT or FD-20 °C for up to a year.     

Influence of common preanalytical variations on the metabolic profile of serum samples in biobanks

A blood pre-centrifugation delay of 24 h at room temperature influenced the proton NMR spectroscopic profiles of human serum. A blood pre-centrifugation delay of 24 h at 4°C did not influence the spectroscopic profile as compared with 4 h delays at either room temperature or 4°C. Five or ten serum freeze–thaw cycles also influenced the proton NMR spectroscopic profiles. Certain common in vitro preanalytical variations occurring in biobanks may impact the metabolic profile of human serum.     

Effect of storage time and temperature on steroid and protein hormone concentrations in porcine plasma and serum

The effect of storage time and temperature of porcine blood prior to quantitation of hormone concentrations by radioimmunoassay (RIA) was evaluated. Blood from each of four luteal phase gilts was used to determine cortisol (CS) and progesterone (P) concentrations, while blood from each of four ovariectomized gilts and each of four lactating sows was used to determine luteinizing hormore (LH) and prolactin (PRL) concentrations, respectively. Blood was collected via jugular puncture from each animal within a 30-sec time period and placed into 18 heparinized and 18 nonheparinized tubes. One sample with and without heparin was stored in ice water (4°C) or at 28°C for 0.25, 2, 4, 6, 8, 12, 24, 36 or 48 hours. After storage, blood was centrifuged at 4°C and plasma or serum was collected and stored at ?20°C until quantitated by RIA. There were no differences (P>0.05) between plasma and serum concentrations (X ± SE, ng/ml) of CS (26.9 ± 0.8 vs 28.5 ± 0.8), P (24.7 ± 0.7 vs 24.8 ± 0.8), LH (2.1 ± 0.1 vs 2.2 ± 0.1) or PRL (53.2 ± 2.3 vs 52.6 ± 2.1). Similarly, storage temperature (4 vs 28°C) did not affect the concentrations of P (25.7 ± 0.8 vs 23.9 ± 0.7), LH (2.2 ± 0.1 vs 2.2 ± 0.1) or PRL (53.7 ± 2.1 vs 53.2 ± 2.3). Howver, CS concentrations decreased (P<0.05) from 28.5 ± 0.5 (4°C) to 26.9 ± 0.8 ng/ml (28°C). There was an animla x time interaction for CS concentration when plasma and serum were stored at both 4°C (P<0.001) and 28°C (P<0.003). There was also and animal x time interaction (P<0.03) for LH concentrations. The P and PRL concentrations decreased linearly by 0.0615 ng/hr (P<0.001) and 0.0625 ng/hr (P<0.004), respectively, with increased storage time.     

Measurement of bisphenol A in human urine using liquid chromatography with multi-channel coulometric electrochemical detection

Smith-Lemli-Opitz syndrome (SLOS) patients have increased 7- and 8-dehydrocholesterol (DHC) concentrations. Using gas chromatography-mass spectrometry with selected ion monitoring we investigated whether storage time (24 h, 7 and 30 days, and 22 months at room temperature or at 4 degrees C) affected DHC concentrations in whole blood spots (WBSs) from SLOS patients and normal controls. Our results suggest that WBS sterol analysis can be used for SLOS screening and possibly related inborn errors of sterol metabolism with a 100% sensitivity and specificity on specimens stored for up to 30 days, either at room temperature or 4 degrees C. After 22 months of storage at both temperature SLOS samples can be indistinguishable from control samples. Therefore, great caution should be used to exclude SLOS by sterol analysis of WBSs stored for a long time.     

Hypoxanthine and xanthine levels determined by high-performance liquid chromatography in plasma, erythrocyte, and urine samples from healthy subjects: the problem of hypoxanthine level evolution as a function of time

The levels of hypoxanthine and xanthine are determined in plasma, erythrocyte, and urine samples by a reverse-phase high-performance liquid chromatographic (HPLC) method. The hypoxanthine concentration increases in erythrocyte and plasma samples when whole blood is stored at room temperature between sampling and centrifugation. Furthermore, the hypoxanthine concentration increases in erythrocyte samples when they are kept apart at room temperature before analysis, whereas the plasma hypoxanthine level remains constant. This result proves an endogenous formation of hypoxanthine in erythrocytes with time, at room temperature. These studies show the necessity of rigorous conditions for the collection, transport, and treatment of blood samples. In order to achieve accurate results, the blood must be centrifuged immediately after collection. The erythrocyte and plasma samples must be stored frozen until deproteinization and HPLC analysis. Under these conditions, the concentrations of hypoxanthine and xanthine in plasma are 2.5 +/- 1 and 1.4 +/- 0.7 microM, respectively. In erythrocyte samples, hypoxanthine concentration reaches 8.0 +/- 6.2 microM.     

Administration of heparin causes in vitro release of non-esterified fatty acids in human plasma

The objective of this study was to investigate the extent to which $\text{in vitro}$ hydrolysis of endogenous triglycerides contributes to the elevated concentrations of non-esterified fatty acids (NEFAs) which have been reported after heparin administration. Heparin is known to induce the release of lipases which hydrolyze endogenous substrate both $\text{in vivo}$ and $\text{in vitro}$. Four patients undergoing diagnostic cardiac catheterization, who routinely receive heparin, were studied. Blood samples were obtained before and at 5 and 30 minutes after an intravenous bolus of heparin (46 U/kg) was administered. Determinations of NEFAs in plasma were carried out immediately and again various times after the samples had incubated at 24°C and at 0°C. In addition, an aliquot of each sample was frozen quickly, stored for 5–7 days, thawed, and incubated at 24°C for 180 minutes. As expected, there were no significant increases after incubation in the concentrations of NEFAs in the samples obtained before heparin administration. In contrast, in the samples obtained after heparin administration, incubation at 24°C produced significant increases in the concentrations of NEFAs. For example, in the plasma samples obtained 5 minutes after administration of heparin, concentrations of NEFAs increased 50, 160 and 300% after 5, 60, and 180 minutes of incubation compared to pre-heparin concentrations. When assayed immediately, the concentrations of NEFAs increased only 15% over pre-heparin concentrations. Incubating the samples at 0°C slowed lipase activity. Freezing the samples stopped the lipase activity; however, when the thawed samples were incubated at 24°C, concentrations of NEFAs continued to rise. This study suggests that much of the reported increases in the $\text{in vivo}$ concentrations of NEFAs after administration of heparin may be due to $\text{in vitro}$ formation from continued lipase activity on endogenous substrate. Moreover, studies relating increases in the concentrations of NEFAs after administration of heparin to changes in drug binding to plasma proteins should be re-examined for possible $\text{in vitro}$ artifacts.     

Preservation of nucleic acid integrity in guanidine thiocyanate lysates of whole blood

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4°C, and more than a year at ?20°C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.     

Effects of storage conditions on the stability and distribution of clinical trace elements in whole blood and plasma: Application of ICP-MS

BackgroundKnowledge of trace element stability during sample handling and preservation is a prerequisite to produce reliable test results in clinical trace element analysis.MethodAn alkaline dissolution method has been developed using inductively coupled plasma mass spectrometry to quantify eighteen trace element concentrations: vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, selenium, bromine, molybdenum, cadmium, antimony, iodine, mercury, thallium, lead, and bismuth in human blood, using a small sample volume of 0.1 mL. The study evaluated the comparative effects of storage conditions on the stability of nutritionally essential and non-essential elements in human blood and plasma samples stored at three different temperatures (4 °C, −20 °C and −80 °C) over a one-year period, and analysed at multiple time points. The distribution of these elements between whole blood and plasma and their distribution relationships are illustrated using blood samples from 66 adult donors in Queensland.ResultsThe refrigeration and freezing of blood and plasma specimens proved to be suitable storage conditions for many of the trace elements for periods up to six months, with essentially unchanged concentrations. Substantially consistent recoveries were obtained by preserving specimens at −20 °C for up to one year. Ultra-freezing of the specimens at −80 °C did not improve stability; but appeared to result in adsorption and/or precipitation of some elements, accompanied by a longer sample thawing time. A population sample study revealed significant differences between the blood and plasma concentrations of six essential elements and their relationships also varied significantly for different elements.ConclusionBlood and plasma specimens can be reliably stored at 4 °C for six months or kept frozen at −20 °C up to one year to obtain high quality test results of trace elements.     

Effect of time and temperature on bovine serum and plasma progesterone concentration

Whole blood, with and without anticoagulant, from 5 pregnant cows was incubated at 40°C for 0 (30 minutes after collection), 6 and 24 hours (hr) before the blood was centrifuged and the plasma or serum was frozen for later progesterone assay. Mean plasma progesterone concentration decreased from 6.6 ng/ml at 0 hr to 1.7 ng/ml at 6 hr (P < 0.01) and to 2.8 ng/ml at 24 hr (P < 0.01). Mean serum progesterone concentration decreased from 6.1 ng/ml at 0 hr to 3.9 ng/ml at 6 hr (P < 0.01) and to 4.4 ng/ml at 24 hr (P < 0.01). Whole blood samples with and without EDTA were also incubated at 4°C for 24 hr. Mean plasma progesterone concentration decreased from 6.6 ng/ml at 0 hr to 4.2 ng/ml at 24 hr (P < 0.01). Mean serum progesterone concentration decreased from 6.1 ng/ml at 0 hr to 4.7 ng/ml at 24 hr (P < 0.01). The incubation time and temperature of whole blood, from collection of blood to the separation of serum or plasma, significantly affects assayable concentration of progesterone.     

High-performance liquid chromatographic assay for meropenem in serum

High-performance liquid chromatographic procedures have been developed for the measurement of meropenem in serum. The separation was performed on an Ultrasphere XL-ODS analytical column (75×4.6 mm I.D.). The mobile phase consisted of 10.53 mmol/l ammonium acetate-acetonitrile (95:5, v/v) (pH 4). The UV detection was at 298 nm. The quantitation limit both in serum and water was 0.25 μg/ml. The method was validated in serum and aqueous solution over the concentration range 0.25–50 μg/ml. The extraction recovery from serum spiked with meropenem was 99.7±3.4%. The intra- and inter-assay coefficients of variation were below 6%. Stored at −80°C for three months at various concentrations in serum and in aqueous solution, meropenem did not reveal any appreciable degradation. After 24 h, it was also stable at 4°C in serum, aqueous solution and supernatant of extraction but not at room temperature. The stability of the drug was also confirmed in serum after repeated freezing-thawing cycles at −80°C on four consecutive days.     

Effect of exercise-induced hyperthermia on serum iron concentration

Serum iron levels have been shown to decline both with fever and with strenuous exercise, leading to the supposition that the decrease might be the result of a rise in core body temperature. To evaluate this hypothesis, the serum iron response to an exercise-induced 1.5°C rise in core body temperature was measured. To increase core temperature, five females and two males exercised in an environmental chamber heated to 41°C with a relative humidity of 40%. Blood samples were taken before exercise and immediately after body temperature increased approximately 1.5°C. Blood was also collected 1 h, 6 h, and 24 h postexercise. Results showed that the core body temperature significantly increased (p<0.001) from a mean baseline value of 36.5±0.1°C to 38.1±0.1°C following exercise. A one-way repeated measures analysis of variance was used to examine the effect of increased core body temperature on serum iron levels over the five time periods: preexercise, immediate postexercise, and 1 h, 6 h, and 24 h postexercise. The results indicated that there were no significant differences in serum iron levels among time periods. This suggests that the previously reported depression of serum iron levels that occurs with fever and after prolonged exercise is not the result of hyperthermia. Rather, the change in serum iron occurs in response to biological or physiological stressors, such as bacterial infection, muscle damage, or unusual trauma. Further studies are needed to explicate the mechanisms responsible for these changes.