Serotonin Secretion by Blood Platelets: Accuracy of High-performance Liquid Chromatography-electrochemical Technique Compared with the Isotopic Test and Use in a Clinical Laboratory

Overview

Journal Res Pract Thromb Haemost

Publisher Elsevier

Date 2023 Aug 21

PMID 37601022

Authors

Eduardo Aranda

Seiki Iha

Sandra Solari

David Rodriguez

Viviana Romero

Luis Villarroel

Jaime Pereira

Olga Panes

Diego Mezzano

Affiliations

Soon will be listed here.

Abstract

Background: Mild secretion defects are the most frequent and challenging blood platelet disorders to diagnose. Most δ-granule secretion tests lack validation, are not quantitative, or have unreliable response to weak platelet agonists.

Objectives: To compare platelet serotonin secretion by HPLC-electrochemical detection technique (HPLC-ECD) with the reference isotopic test (H-5-HT), evaluating its performance in clinical laboratories.

Methods: The assay validation followed STARD-2015 recommendations. HPLC-ECD measured the nonsecreted serotonin remaining in platelet pellets after aggregation, comparing it with the reference H-5-HT assay. We studied subjects with inherited and aspirin-induced blood platelet disorders and assessed the HPLC-ECD operation for routine clinical diagnosis.

Results: Calibration curves were linear (R = 0.997), with SD for residuals of 3.91% and analytical sensitivity of 5ng/mL. Intra- and interassay imprecision bias ranged between -8.5% and 2.1% and -9% and 3.1%, respectively. Serotonin recovery and stability were >95%, and the variability range of measurements was -5.5% to 4.6%. Statistical differences detected between tests were biologically irrelevant, with bias of 1.48% (SD, 8.43) and CI agreement of -18% to 15%. Both assays distinctly detected platelet secretion induced by 10 μM epinephrine and 4 μmM adenosine diphosphate. However, HPLC-ECD is quantitative and more sensitive to low serotonin content in blood platelets. Reference cutoffs for each agonist were determined in 87 subjects. Initially, the HPLC-ECD requires relatively expensive equipment and trained operators but has remarkably cheap running costs and a turn-around time of 24-36 hours. We have used this diagnostic tool routinely for >8 years.

Conclusion: HPLC-ECD assay for platelet serotonin secretion is highly accurate, has advantages over the reference H-5-HT test, and is suitable as a clinical laboratory technique.

References

Fouassier M, Babuty A, Debord C, Bene M . Platelet immunophenotyping in health and inherited bleeding disorders, a review and practical hints. Cytometry B Clin Cytom. 2020; 98(6):464-475. DOI: 10.1002/cyto.b.21892. View

Lotta L, Maino A, Tuana G, Rossio R, Lecchi A, Artoni A . Prevalence of disease and relationships between laboratory phenotype and bleeding severity in platelet primary secretion defects. PLoS One. 2013; 8(4):e60396. PMC: 3614926. DOI: 10.1371/journal.pone.0060396. View

Aranda E, Pereira J, Ajenjo C, Prieto C, Sepulveda S, Mezzano D . Human intraplatelet 5-hydroxytryptamine is correlated with mean platelet survival time. Thromb Res. 1996; 84(1):67-72. DOI: 10.1016/0049-3848(96)00162-4. View

Quiroga T, Goycoolea M, Panes O, Aranda E, Martinez C, Belmont S . High prevalence of bleeders of unknown cause among patients with inherited mucocutaneous bleeding. A prospective study of 280 patients and 299 controls. Haematologica. 2007; 92(3):357-65. DOI: 10.3324/haematol.10816. View

Mombouli J, Vanhoutte P . Endothelial dysfunction: from physiology to therapy. J Mol Cell Cardiol. 1999; 31(1):61-74. DOI: 10.1006/jmcc.1998.0844. View

Mumford A, Nisar S, Darnige L, Jones M, Bachelot-Loza C, Gandrille S . Platelet dysfunction associated with the novel Trp29Cys thromboxane A₂ receptor variant. J Thromb Haemost. 2013; 11(3):547-54. DOI: 10.1111/jth.12117. View

Holmsen H, Day H, Setkowsky C . Secretory mechanisms. Behaviour of adenine nucleotides during the platelet release reaction induced by adenosine diphosphate and adrenaline. Biochem J. 1972; 129(1):67-82. PMC: 1174042. DOI: 10.1042/bj1290067. View

Kumar A, Kumar M, Deepika K, Fernandez J, Eisdorfer C . A modified HPLC technique for simultaneous measurement of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in cerebrospinal fluid, platelet and plasma. Life Sci. 1990; 47(19):1751-9. DOI: 10.1016/0024-3205(90)90349-v. View

Kambayashi J, Shinoki N, Nakamura T, Ariyoshi H, Kawasaki T, Sakon M . Prevalence of impaired responsiveness to epinephrine in platelets among Japanese. Thromb Res. 1996; 81(1):85-90. DOI: 10.1016/0049-3848(95)00216-2. View

10.

Drummond A, Gordon J . Letter: Rapid, sensitive microassay for platelet 5HT. Thromb Diath Haemorrh. 1974; 31(2):366-7. View

11.

Cattaneo M, Cerletti C, Harrison P, Hayward C, Kenny D, Nugent D . Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH. J Thromb Haemost. 2013; . DOI: 10.1111/jth.12231. View

12.

Matus V, Valenzuela G, Saez C, Hidalgo P, Lagos M, Aranda E . An adenine insertion in exon 6 of human GP6 generates a truncated protein associated with a bleeding disorder in four Chilean families. J Thromb Haemost. 2013; 11(9):1751-9. DOI: 10.1111/jth.12334. View

13.

Wall J, Arnold J, Wang W, White M, Jennings L, Jackson C . A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents. Br J Haematol. 1995; 89(2):380-5. DOI: 10.1111/j.1365-2141.1995.tb03315.x. View

14.

Pai M, Wang G, Moffat K, Liu Y, Seecharan J, Webert K . Diagnostic usefulness of a lumi-aggregometer adenosine triphosphate release assay for the assessment of platelet function disorders. Am J Clin Pathol. 2011; 136(3):350-8. DOI: 10.1309/AJCP9IPR1TFLUAGM. View

15.

Holmsen H, Day H, Stormorken H . The blood platelet release reaction. Scand J Haematol Suppl. 1969; 8:3-26. View

16.

Lages B, Weiss H . Heterogeneous defects of platelet secretion and responses to weak agonists in patients with bleeding disorders. Br J Haematol. 1988; 68(1):53-62. DOI: 10.1111/j.1365-2141.1988.tb04179.x. View

17.

Feinman R, Lubowsky J, Charo I, Zabinski M . The lumi-aggregometer: a new instrument for simultaneous measurement of secretion and aggregation by platelets. J Lab Clin Med. 1977; 90(1):125-9. View

18.

Mezzano D, Aranda E, Rodriguez S, Foradori A, Lira P . Increase in density and accumulation of serotonin by human aging platelets. Am J Hematol. 1984; 17(1):11-21. DOI: 10.1002/ajh.2830170103. View

19.

Mumford A, Frelinger 3rd A, Gachet C, Gresele P, Noris P, Harrison P . A review of platelet secretion assays for the diagnosis of inherited platelet secretion disorders. Thromb Haemost. 2015; 114(1):14-25. DOI: 10.1160/TH14-11-0999. View

20.

Mezzano D, Harrison P, Frelinger 3rd A, Mumford A, Noris P, Lordkipanidze M . Expert opinion on the use of platelet secretion assay for the diagnosis of inherited platelet function disorders: Communication from the ISTH SSC Subcommittee on Platelet Physiology. J Thromb Haemost. 2022; 20(9):2127-2135. DOI: 10.1111/jth.15781. View