Date of Award
Master of Science
R. L. Murphree
D. O. Richardson, J. B. McLaren
Competitive protein binding (CPB) assay for progesterone and radioimmunoassay (RIA) for estrogen were used to evaluate the effects of various blood handling techniques from the time of collection to assay on the stability of progesterone and estrogen concentrations in blood. Eight 40 ml samples were collected consecutively in polycarbonate centrifuge tubes from the jugular veins of eight cows and subjected to various treatments. Treatments consisted of collecting samples in pre-cooled heparinized (1000 U/ml) tubes maintained in an ice bath, centrifuging immediately (2400 rpm for 30 minutes) and storing the plasma fraction at -15°C; and collecting samples in heparinized or non-heparinized, pre-cooled and warm (room temperature) tubes, and holding as whole blood either at 4°C for 24 hours or at room temperature for 4 or 24 hours prior to centrifutation and storage of the plasma or serum fraction at -15°C. The last of the eight samples collected from.each cow was handled identically to the first sample to evaluate the effects of animal stress during bleeding and volume of blood collected on hormone concentration. Significant differences in the progesterone concentrations existed between treatments (P < 0.001). Plasma and serum samples held at 4°C for 24 hours were significantly lower in progesterone than plasma samples centrifuged. There was no difference in progesterone concen-trations of plasma and serum samples subjected to similar treatment effects. Samples collected in warm (room temperature) tubes and held as whole blood at room temperature until centrifuged were significantly lower in progesterone content (P < 0.001) than those collected in cool tubes. Warm tube samples held as whole blood at room temperature for 24 hours prior to centrifugation were significantly lower in progesterone than those held as whole blood for 4 hours (P < 0.001). Serum samples held at room temperature for 4 hours and 24 hours prior to centrifugation were 38 percent and 89 percent lower in progesterone content, respectively, than the plasma samples centrifuged immediately. Animal stress or volume of blood collected had no effect on progesterone concentration. Radioimmunoassay for estrogen was conducted on blood from the same cows and subjected to the same treatments used for CPB assay of progesterone. There was no significant difference in estrogen concen-trations between any of the treatments evaluated in this study. Aliquots of pooled plasma samples from a number of cows were subjected to alternate freezing and thawing to a maximum of 6 and 7 times prior to the assay for progesterone and estrogen, respectively. No significant effect of repeated freezing and thawing on the concentra-tion of either progesterone or estrogen was detected. The average recovery of labeled progesterone added to cow plasma was 93.3 ± 0.64 percent. Following the addition of 3 ng of unlabeled progesterone to steer plasma, the average recovery was 96.9 ± 0.45 per cent or 2.9 ng of progesterone. RIA was carried out using one 6 ml extraction and 2 hours lee water Incubation. Recovery of 20 pg and 50 pg of estradiol-17β added to pooled cow plasma averaged 91.0 ± 0.35 per cent and 81.0 ± 0.60 percent, respectively. Average recovery of estradiol-17β3H from a cow being assayed was 89.0 ± 1.5 percent. Based on the data obtained in this study, samples used for CPE assay of progesterone should be centrifuged and the plasma fraction of whole blood removed as soon as possible after collection and frozen until assayed. Estrogen concentrations in samples prepared for RIA of estro-gen are apparently not as susceptible to the effects of various sample preparation methods as is progesterone. However, it is recommended that samples of estrogen assay also be centrifuged and stored at sub zero temperatures as soon as possible.
Wenzel, Vaughan A., "The effects of blood preparation for radioimmunoassay and competitive protein binding assay on the stability of progesterone and estrogen concentrations. " Master's Thesis, University of Tennessee, 1972.