Pre-analytical factors affecting measurement of plasma kisspeptin by radioimmunoassay
Radha Ramachandran, Michael Patterson, Kevin G Murphy, Waljit S Dhillo, S Patel, A Kazarian, Mohammad A Ghatei & Stephen R Bloom
Background: Kisspeptin is a peptide product of the KiSS-1 gene and a key regulator of the hypothalamo-pituitary-gonadal axis. Pregnancy is associated with raised plasma kisspeptin concentrations. However, there is significant variation in the plasma kisspeptin levels quoted in previous studies. We investigated the effects of pre-analytical factors including processing time and anticoagulant type on plasma kisspeptin-immunoreactivity (IR) measurement.
Method: Following ethical approval and informed consent, 1 ml of blood from four pregnant women was collected into each of five tube types: serum clot-activator (S), serum separator (SST), lithium heparin with 200 ul/tube of trasylol (L), citrate (C), and ethylenediaminetetraacetic acid (E) tubes. All tubes were centrifuged and serum or plasma separated at t=0, 1 h, 2 h, and 4 h post-collection and stored at −80 °C. Kisspeptin-IR in all samples was measured by a specific radioimmunoassay in a single assay.
Results: Kisspeptin-IR was poorly preserved in serum samples, but was relatively stable in plasma. There was no statistically significant difference in kisspeptin-IR from samples collected in E, L or C tubes, though samples collected in C tubes consistently contained lower kisspeptin-IR concentrations. Kisspeptin-IR levels decreased with increased processing times. Highest levels of kisspeptin-IR were obtained from samples processed immediately. Freeze-thaw cycles did not significantly influence plasma kisspeptin-IR levels.
Discussion: These results suggest that E or lithium heparin tubes are the most suitable for the collection of human plasma samples for measurement of kisspeptin-immunoreactivity. Blood samples should be immediately separated. However, repeated freeze-thaw cycles have no significant effect on measurement of plasma kisspeptin-immunoreactivity.