Very Long-Term Stability of Tryptase in Frozen Serum Samples

Abstract

Mast cells are key players in allergic manifestations, which will affect 50% individuals by 2050. In physiological conditions, tryptase is produced almost exclusively by mast cells. A transient serum tryptase elevation is the biological hallmark of systemic acute mast cell activation, for example, occurring during anaphylaxis or Mast Cell Activation Syndrome.

In contrast, basal serum tryptase (bST) is remarkably steady over years in a given individual [1]. The concentrations of bST are mainly determined by genetic factors like Hereditary alpha-Tryptasemia [1, 2] and by the number of mast cells, which evolves very slowly in most healthy and diseased individuals [1]. Inter-individual variability of bST is explained by several sociodemographic, environmental and health determinants [3]. Notably, we recently reported that higher bST concentrations were associated with higher risk of allergic sensitisation, FeNO values, allergic manifestations including IgE-mediated asthma, and poor asthma control, in teenagers from a birth cohort [4]. Given the lifelong intraindividual stability of bST, our results suggest that bST could be used as a predictive tool to stratify individuals at high risk of allergic manifestations. Provided sufficient tryptase stability in frozen biological samples, this hypothesis could be tested retrospectively on research and care setting biobanks. Long-term tryptase stability is highly suspected [1] but was never formally demonstrated to date.

The present study aimed to determine very long-term tryptase stability in a collection of serum samples frozen during several years.

Briefly, all samples were collected at the Armand-Trousseau (AP-HP) hospital allergology laboratory from January to December 2016 (T0), systematically frozen and stored at −20°C immediately after bST measurements, and reanalysed for bST in July 2024 (T8). Blood samples were collected on tubes with clot activator and send to the laboratory within 2 h at room temperature. After 2 h clotting, samples were centrifuged at 1500 g, 15°C. Sera were aliquoted, stored at +4°C, and bST was analysed within 7 days [5]. If enough sample remained, sera were stored right after bST measurement at −20°C until T8. The maintenance of adequate temperature during storage was monitored by a temperature probe (SPY RF, JRI (France)) connected to a live monitoring software (Sirius, JRI (France)). Samples were kept at −20°C (±4°C) during all the study, without major incident or defrosting. A total of exactly 100 samples with bST values at T0 within reference range (1–15 μg/L) [1] were retrieved. Samples were allowed to defrost in an upright position during 24 h at +4°C. After defrosting, sera were homogenised gently by a vortex at low intensity and centrifuged before reanalysis. Tryptase were re-assayed at T8 by the same method (ImmunoCAP Tryptase Test, ThermoFisher Scientific, Uppsala, Sweden), and on the same instrument (Phadia250). All tests were performed after appropriate calibration and internal controls and according to the manufacturer's recommendations.

The bST assessed at T8 showed good correlation with the measure made at T0 (Spearman's rho = 0.86). Data fitted well a log–log linear model (see Figure 1). The intercept estimate was b = −0.024 (95% confidence interval [−0.094; 0.046]), and the slope estimate was a = 0.935 (95% CI [0.834; 1.036]). Overall, this model did not differ significantly from the null model (b = 0 and a = 1). The fulfilment of assumptions of linear regression models (e.g., the independence of observations, the normal distribution of residuals, homoscedasticity) was checked using inspection of Residuals distribution, Residuals Vs Fitted, Q-Q plot, Scale-Location and Residuals Vs Leverage plots. We conclude that bST is very stable in frozen serum samples and can be accurately measured years after sampling, if conserved the appropriate way.

Over the years, tryptase has overshadowed histamine as the main mast cell biomarker, partly because of its longer half-life in vivo, later demonstrated ex vivo in whole blood samples [5]. Because tryptase is not routinely performed on a daily basis in most laboratories, specimens are usually frozen at −20°C until analysis. To the best of our knowledge, the present study is the first to evaluate tryptase stability in frozen serum samples. Results from assays measuring enzymatic activity tend to decrease even after 1 month at −20°C [6]. However, tryptase concentrations are measured by a fluoroenzymatic immunoassay which is independent of its enzymatic activity. To date, this is the only commercial method used in clinical setting.

Hence, these results would apply in any clinical laboratory measuring tryptase on frozen samples and can serve as documentation for quality requirements. In addition, our study provides the conceptual ground for retrospective tryptase measurement on frozen samples from various cohorts, even many years after sampling.

In conclusion, we show that tryptase is very stable and can be measured reliably on serum samples frozen for years.

Y.C. proposed the concept of the letter and designed the study. F.B. and T.R. contributed to data collection. S.B. and B.B. contributed to samples analysis. F.B. and Y.C. performed search of the literature and wrote the manuscript. Y.C. performed the statistical analysis and provided revisions to the draft. Y.C. and F.B. interpreted the results. D.K.T., A.A. and S.C. helped with manuscript review. All authors have reviewed the final draft and given their final approval for submission.

Y. Chantran serves in scientific advisory board for ThermoFisher Scientific, received honoraria from ThermoFisher Scientific and research grants from Blueprint Medicines and ThermoFisher Scientific. The rest of the authors declare that they have no relevant conflicts of interest.