️ Episode 152: One-Well Multiplex ddPCR for Hereditary Alpha Tryptasemia

In this episode of PaperCast Base by Base, we explore a validated single‑well multiplex digital droplet PCR (ddPCR) assay that reconstructs the TPSAB1 locus by quantifying α‑ and β‑tryptase copy numbers to diagnose hereditary alpha tryptasemia (HαT) in symptomatic patients.

Study Highlights:
The authors designed a triplex ddPCR assay that simultaneously measures α‑ and β‑tryptase copy numbers and an internal reference in one well, avoiding cross‑reactivity and excessive signal “rain” while achieving clear cluster separation and straightforward thresholds. In analytical validation across 281 cases, copy‑number calls tightly clustered around expected integers with an overall 99% prediction interval of 0.03 ± 0.27 copies and showed 100% concordance with a prior double‑well reference assay for all genotypes tested. In a clinical subcohort of 141 symptomatic patients without other causes of elevated tryptase, basal serum tryptase (BST) predicted HαT with an optimal cutoff of 9.2 ng/mL, yielding 98.1% sensitivity and 96.6% specificity. A strong gene–dose relationship was observed, with BST increasing by an average of 7.5 ng/mL for each additional α‑tryptase copy, and thresholds helped distinguish duplications from higher‑order α‑copy states.

Conclusion:
A simple, robust single‑well multiplex ddPCR enables accurate HαT genotyping and provides actionable BST thresholds to triage symptomatic patients for confirmatory testing.

Reference:
Alheraky A, Wierenga ATJ, Simpelaar A, Hesp LB, Minovic I, Bagheri N, Roozendaal C, Span LFR, Oude Elberink HNG, Kema IP, Mulder AB. Hereditary Alpha Tryptasemia: Validation of a Single‑Well Multiplex Digital Droplet PCR Assay in a Cohort of Symptomatic Patients. Clinical Chemistry. 2024;70(2):425–433. https://doi.org/10.1093/clinchem/hvad206

License:
This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International License (CC BY 4.0) – https://creativecommons.org/licenses/by/4.0/

Support:
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Episode Slug: one-well-multiplex-ddpcr-for-hereditary-alpha-tryptasemia

Keywords: hereditary alpha tryptasemia; TPSAB1 copy number; digital droplet PCR; basal serum tryptase; diagnostic cutoff

Chapters

• (00:00:00 ) - Hereditary erypticemia: Solving the genetic puzzle
• (00:05:05 ) - Mastocytosis DNA test: Single Well Multiplex Digital
• (00:09:18 ) - How did the new single well test perform?
• (00:15:15 ) - Heidelberg Disease: The new test for hereditary alpha trypt