Hereditary Alpha Tryptasemia: Validation of a Single-Well Multiplex Digital Droplet PCR Assay in a Cohort of Symptomatic Patients

Background Hereditary alpha tryptasemia (H alpha T) has significant prevalence and potential morbidity in the general population. However, it remains largely undiagnosed in routine clinical diagnostics due to low availability of efficient assessment methods. To address this issue, we developed a reliable and efficient single-well multiplex digital droplet PCR assay.Methods The assay was based on the reconstruction of the TPSAB1 gene through quantification of the ratio of alpha- and beta-tryptase copy number variants (CNV) in a single-well measurement. We performed analytical validation by determining CNV measurement clustering around the expected copy numbers in 281 cases and determined the diagnostic accuracy of basal serum tryptase (BST) to predict H alpha T and H alpha T subtypes in 141 symptomatic patients.Results The assay determined alpha- and beta-tryptase CNVs with an overall accuracy, expressed as a 99% prediction interval, of 0.03 +/- 0.27 copy numbers. The optimal BST cutoff level to predict H alpha T in symptomatic patients, who had no other explanation for relatively high tryptase levels (i.e., no diagnosis of systemic mastocytosis, myeloid neoplasm, or end-stage renal failure), was 9.2 ng/mL (sensitivity: 98.1%; specificity: 96.6%). H alpha T showed a linear gene-dose effect, with an average gene-dose increase of 7.5 ng/mL per extra alpha-tryptase gene.Conclusion Our single-well multiplex digital droplet PCR assay accurately determined H alpha T and could be implemented as a state-of-the-art routine diagnostic test. The assay demonstrated a strong correlation with BST and the optimal threshold for identifying H alpha T in symptomatic patients with unexplained high tryptase concentrations was at a BST level of 9.2 ng/mL.