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1 Department of Metabolic Medicine, Imperial College London, Hammersmith Hospital, London, UK
2 Division of Clinical Chemistry, Hammersmith Hospitals NHS Trust, London, UK
aAddress correspondence to this author at: Department of Metabolic Medicine, Imperial College London, 6th Floor Commonwealth Building, Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK, Fax +44 (0) 20 8383 3142, e-mail s.bloom{at}imperial.ac.uk
To the Editor:
Kisspeptins are peptide products of the KiSS-1 metastasis-suppressor (KISS1) gene and the natural ligands of the G-protein–coupled receptor GPR54. KISS1 was initially investigated as an antimetastasis gene. More recent studies have demonstrated that the kisspeptins are potent stimulators of the hypothalamo-pituitary-gonadal axis. Mice and humans with defective kisspeptin signaling show hypogonadotrophic hypogonadism and impaired sexual development (1)(2).
Plasma kisspeptin concentrations are <2 pmol/L in men and nonpregnant women. KiSS-1 mRNA is highly expressed in the placenta, and plasma kisspeptin concentrations increase dramatically, to thousands of picomoles per liter, during pregnancy (3). In addition, plasma kisspeptin is increased in women with gestational trophoblastic tumors, thus raising the possibility of measuring plasma kisspeptin as a novel tumor marker (4). Previous studies that have measured plasma kisspeptin in women during pregnancy have found significantly different concentrations of circulating kisspeptin (3)(4) that may be attributable to differences in preanalytical variables, such as collection tube type, processing times, and storage conditions.
Use of a standardized sample collection method for the measurement of circulating kisspeptin immunoreactivity (IR) would facilitate comparisons between separate studies and is necessary if plasma kisspeptin concentrations are to be used as a disease marker. We thus assessed the effects of processing time, anticoagulant type, and repeated freeze-thaw cycles on kisspeptin-IR measurement in plasma samples.
To evaluate the effects of collection tube type and processing time on plasma kisspeptin-IR measurement, we recruited 4 healthy pregnant women who gave informed consent [age range 25–37 years, mean (SD) 32.8 (5.3) years; body mass index (BMI) 19–35.5, 25.4 (7.07); gestation 15–35 weeks, 27 (8.83) weeks]. Blood was collected, using a Vacutainer® system, directly into 4 tubes of each of the tube-types studied: lithium heparin with 2000 U/tube of trasylol (L), citrate (C), EDTA (E), serum clot-activator (S), and serum separator (SST) tubes (BD Vacutainer® Blood Collection Tubes). Both S and SST tubes are coated with silicone and micronized silicon particles to accelerate clotting. The SST tubes have, in addition, a barrier polymer at the bottom of the tube. The density of this polymer allows it to rise up to the clot–serum interface during centrifugation, thus forming a physical barrier separating the serum from the clot.
Of the 4 samples in each tube type, the first was processed immediately, and the second, third, and fourth samples were maintained at room temperature (18–20 °C) for 1, 2, and 4 h, respectively, before processing. All samples were processed by centrifugation at 4 °C for 10 min at 855g. Serum and plasma were then aspirated, divided into aliquots, frozen, and stored at –80 °C until assayed. Kisspeptin-IR was assayed using a specific RIA (5). The detection limit of the assay was 2 pmol/L of plasma kisspeptin-IR at a 95% confidence limit. The inter- and intraassay CVs were <11% and <9%, respectively. All samples were assayed in duplicate. Differences of means were assessed by paired t-test.
Kisspeptin-IR degraded rapidly in serum tubes. Kisspeptin-IR was undetectable in serum samples that were processed at t = 1 h and later time points, and concentrations detected in serum samples processed immediately were significantly lower than those detected in plasma (P <0.05). Kisspeptin-IR concentrations in plasma samples collected in C tubes were consistently lower than those obtained from E and L collected samples, but this difference was not statistically significant (Table 1
). Kisspeptin-IR in plasma samples decreased with increased processing time, suggesting that kisspeptin-IR is best measured in plasma samples that are processed immediately after sampling.
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To evaluate the effects of repeated freezing and thawing on plasma kisspeptin-IR concentrations, 5 mL of blood from each of 4 volunteers [age range 23–41 years, mean (SD) 30.5 (7.6) years; BMI 22–28, 25 (2.45); gestation 23–31 weeks, 30 (0.82) weeks] was collected directly into L and E collection tubes. L and E tubes were used, on the basis of results obtained in the first part of the study, which suggested that kisspeptin-IR was most stable in these 2 tube types. The samples were centrifuged and plasma was separated immediately after collection. An aliquot was taken from the plasma before the first cycle of freezing and after 1, 2, and 3 freeze-thaw cycles. Kisspeptin-IR concentrations in samples collected in L or E tubes did not significantly change even after 4 freeze-thaw cycles (data not shown).
Our studies suggest that circulating kisspeptin-IR concentrations should be measured in plasma samples processed immediately after collection. L or E tubes may preserve kisspeptin-IR better than C tubes. Repeated freezing and thawing does not significantly influence the measurement of kisspeptin-IR concentrations.
Acknowledgments
Grant/funding Support: KGM is supported by a BBSRC New Investigator Award. MP is supported by the BBSRC. WSD is supported by a UK Department of Health Clinician Scientist Award. The Department of Metabolic Medicine receives funding from the Medical Research Council, Wellcome Trust, EU 6th Framework Programme (LSHM-CT-2003-503041) and the NIHR Biomedical Research Centre Funding Scheme.
Financial Disclosures: None declared.
References
The following articles in journals at HighWire Press have cited this article:
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  R. M Reynolds, J. J Logie, A. K Roseweir, A. J McKnight, and R. P Millar A role for kisspeptins in pregnancy: facts and speculations Reproduction, July 1, 2009; 138(1): 1 - 7. [Abstract] [Full Text] [PDF]
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