Primate Monocytes - CD14, CD16 - Ziegler-Heitbrock

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

Abstract

BACKGROUND: Hereditary alpha tryptasemia (HalphaT) 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 HalphaT and HalphaT 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 HalphaT 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%). HalphaT 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 HalphaT 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 HalphaT in symptomatic patients with unexplained high tryptase concentrations was at a BST level of 9.2 ng/mL.