Preanalytical Factors Affecting RIA Measurement of Plasma Kisspeptin (original) (raw)

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Department of Metabolic Medicine

, Imperial College London, Hammersmith Hospital, London, UK

Division of Clinical Chemistry

, Hammersmith Hospitals NHS Trust, London, UK

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Department of Metabolic Medicine

, Imperial College London, Hammersmith Hospital, London, UK

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Department of Metabolic Medicine

, Imperial College London, Hammersmith Hospital, London, UK

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Department of Metabolic Medicine

, Imperial College London, Hammersmith Hospital, London, UK

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Department of Metabolic Medicine

, Imperial College London, Hammersmith Hospital, London, UK

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Department of Metabolic Medicine

, Imperial College London, Hammersmith Hospital, London, UK

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Department of Metabolic Medicine

, Imperial College London, Hammersmith Hospital, London, UK

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Department of Metabolic Medicine

, Imperial College London, Hammersmith Hospital, London, UK

Address 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@imperial.ac.uk

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Radha Ramachandran, Michael Patterson, Kevin G Murphy, Waljit S Dhillo, Sejal Patel, Anna Kazarian, Mohammad A Ghatei, Stephen R Bloom, Preanalytical Factors Affecting RIA Measurement of Plasma Kisspeptin, Clinical Chemistry, Volume 54, Issue 3, 1 March 2008, Pages 615–617, https://doi.org/10.1373/clinchem.2007.093005
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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 855_g_. 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.

Table 1.

Kisspeptin-IR values (pmol/L) measured in plasma and serum samples collected in 4 different tube types from 4 healthy pregnant women.1

1

The paired _t_-test was used to look for significant differences for a given sample type at different times compared to time zero (P values shown in the final column). Paired _t_-test was used to compare the values at time zero in each tube type to those obtained with lithium heparin tubes (used in our laboratory as a calibrator). At time zero a statistically significant difference was observed between lithium heparin and serum and SST tubes (P <0.05) but not between lithium heparin and EDTA or between lithium heparin and citrate tubes (_P_ >0.05). P = 0.051 for comparison of concentrations obtained in the EDTA and citrate tubes. Kisspeptin-IR concentrations reported to the nearest 100 pmol/L.

Table 1.

Kisspeptin-IR values (pmol/L) measured in plasma and serum samples collected in 4 different tube types from 4 healthy pregnant women.1

1

The paired _t_-test was used to look for significant differences for a given sample type at different times compared to time zero (P values shown in the final column). Paired _t_-test was used to compare the values at time zero in each tube type to those obtained with lithium heparin tubes (used in our laboratory as a calibrator). At time zero a statistically significant difference was observed between lithium heparin and serum and SST tubes (P <0.05) but not between lithium heparin and EDTA or between lithium heparin and citrate tubes (_P_ >0.05). P = 0.051 for comparison of concentrations obtained in the EDTA and citrate tubes. Kisspeptin-IR concentrations reported to the nearest 100 pmol/L.

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.

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

1

Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Jr, Shagoury JK, et al. The GPR54 gene as a regulator of puberty.

N Engl J Med

2003

;

349

:

1614

-1627.

2

Kuohung W, Kaiser UB. GPR54 and KiSS-1: role in the regulation of puberty and reproduction.

Rev Endocr Metab Disord

2006

;

7

:

257

-263.

3

Horikoshi Y, Matsumoto H, Takatsu Y, Ohtaki T, Kitada C, Usuki S, et al. Dramatic elevation of plasma metastin concentrations in human pregnancy: metastin as a novel placenta-derived hormone in humans.

J Clin Endocrinol Metab

2003

;

88

:

914

-919.

4

Dhillo WS, Savage P, Murphy KG, Chaudhri OB, Patterson M, Nijher GM, et al. Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and falls during treatment.

Am J Physiol Endocrinol Metab

2006

;

291

:

E878

-E884.

5

Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK, et al. Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males.

J Clin Endocrinol Metab

2005

;

90

:

6609

-6615.

© 2008 The American Association for Clinical Chemistry

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