Serotonin secretion by blood platelets: accuracy of high-performance liquid chromatography-electrochemical technique compared with the isotopic test and use in a clinical laboratory

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 (³H-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 ³H-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 (R² = 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 ³H-5-HT test, and is suitable as a clinical laboratory technique.

Keywords: blood platelets; clinical laboratory techniques; electrochemical technique; platelet aggregation; platelet secretion; serotonin.

Figure 1

Comparison of ³H-5-HT and high-performance liquid chromatography-electrochemical detection (HPLC-ECD) assays for platelet 5-HT secretion for all the study subjects, those after aspirin intake, and patients with inherited platelet secretion defects (IPSDs). (A) The 5-HT secretion by ³H-5-HT and HPLC-ECD of all the 5-HT measurements in the study, including the normal and abnormal results (n = 730 paired samples), containing a wide range of platelet secretion results observed in the laboratory. The values are strikingly similar, and the statistical difference between both tests probably lacks physiological relevance. (B) Deming linear regression of data as demonstrated in (A). The slope is close to 1.0, Y-intercept is 2.3, the R² coefficient is 0.84, and variance of the residuals Sy.x is 1.13. (C) Comparison of ³H-5-HT and HPLC-ECD tests in subjects after taking aspirin. The statistical difference between both tests at these low, abnormal secretion figures indicates a slightly higher sensitivity of HPLC-ECD over the ³H-5-HT test. (D) A similar comparison was demonstrated in (C) in 14 patients with IPSDs. Some 5-HT secretion values >40% with both methods corresponded to 2 patients with homozygous deficiency of platelet glycoprotein VI, who had normal aggregation with 4 and 8 μM ADP, arachidonate, and also with epinephrine in one of the patients. Further inspections of (C) and (D) suggest that aspirin induces a more severe laboratory defect compared with that in patients with IPSDs. (E) Deming linear regression grouping all the abnormal 5-HT secretion assays (IPSDs and platelets with aspirin effect). Despite the accumulation of values with absent or close to 0% secretion, the slope and Y-intercept are similar to those of (B).

Figure 2

Normal platelet secretion results with ³H-5-HT assay compared with the high-performance liquid chromatography-electrochemical detection (HPLC-ECD) test. Longitudinal consistency of the results with HPLC-ECD. (A) Columns 1 and 2 compare the normal 5-HT secretion by ³H-5-HT testing with the measurements by the HPLC-ECD method (n = 510 paired observations from 87 subjects). Regardless of the statistical significance (Wilcoxon matched-pairs signed rank test), the distribution of values is remarkably similar between both tests and the numerical difference is probably not biologically relevant. The third scatter column shows 4728 normal 5-HT secretion measurements by HPLC-ECD from 788 patients tested between November 2020 and October 2022. These values are not significantly different from the 510 original values measured by ³H-5-HT (column 1). However, the historical HPLC-ECD values are slightly lower than those of column 2, measured also by HPLC-ECD. Again, this small statistical difference is probably physiologically irrelevant. (B) The Bland-Altman plot analysis of the paired results with ³H-5-HT and HPLC-ECD shows a minimal bias with a CI agreement within the predefined range of ±20%. (C) The secretion induced by each agonist with data segregated from columns 1 and 2 in (A). Except for epinephrine- and arachidonate-induced secretion, the differences with the other agonists were not statistically significant between ³H-5-HT and HPLC-ECD tests (paired Student's t-test). The 2.5% percentile represents the cutoff values for each agonist. Epinephrine-induced secretion with both tests is significantly higher than all the other agonists (1-way ANOVA, Dunnet’s multiple comparison test). (D) Similarly, the 4728 5-HT measurements by HPLC-ECD shown in column 3 in (A) were separated and assigned to their respective agonists. The results are similar to the values in (C), except for responses to 4 and 8 μg/mL collagen, which are lower in (D) (P < .0001 and P = .008, Mann–Whitney U-test). Epinephrine-induced secretion in (C) and (D) are almost identical (P = .94, Mann–Whitney U-test). Like in (C), secretion with epinephrine is statistically higher than with all the other agonists (Friedman test, Dunńs multiple comparison test).