{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T17:42:05Z","timestamp":1789062125255,"version":"build-2803163510"},"reference-count":34,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T00:00:00Z","timestamp":1784160000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Detecting a KIT D816V mutation in patients with systemic mastocytosis (SM) needs a high-sensitivity assay. Additionally, high mutant KIT transcriptional activity and multilineage involvement are strong unfavorable prognostic parameters in SM. The current frequently used quantitative PCR-based KIT D816V assays have important analytical limitations, including considerable analytical variation in the low-concentration range, a need for a calibration curve, less multiplexing capabilities, and higher susceptibility to interference by PCR inhibitors. We developed, validated, and explored the diagnostic relevance of a novel multiplex KIT D816V cDNA droplet digital PCR (ddPCR), using ABL1 and KIT as reference genes. Furthermore, we correlated the expressed allele burden (EAB) with variant allele frequency (VAF) and percentage of neoplastic mast cells (nMC%) in bone marrow samples of 79 SM patients. Our assay shows very low background signals and high assay precision, leading to a linear dynamic range spanning five orders of magnitude and very low limits of detection and quantification of 0.062% (EAB\/ABL1) and 0.027% (EAB\/KIT). The assay detects median 57 times more mutant cDNA copies than mutant gDNA copies in SM patients and positivity in 83% of SM-suspected patients with negative gDNA ddPCR results. Furthermore, correlations between EAB, VAF, and nMC% suggest the presence of KIT D816V in non-MCs, either with or without transcriptional activity. Using our novel high-sensitivity mRNA-based multiplex ddPCR KIT D816V assay returns high diagnostic yield in patients with suspicion for SM. We recommend incorporating our assay in routine SM diagnostics to determine low-burden KIT D816V.<\/jats:p>","DOI":"10.3390\/ijms27146314","type":"journal-article","created":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T06:49:36Z","timestamp":1784184576000},"page":"6314","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development of a High-Sensitivity Multiplex mRNA-Based KIT D816V Droplet Digital PCR Assay and Correlation with Tumor Load in Systemic Mastocytosis"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0708-8987","authenticated-orcid":false,"given":"Abdulrazzaq","family":"Alheraky","sequence":"first","affiliation":[{"name":"Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arjan","family":"Simpelaar","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucy B.","family":"Hesp","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7464-850X","authenticated-orcid":false,"given":"Albertus T. J.","family":"Wierenga","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Moniek","family":"Heddema","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5439-3781","authenticated-orcid":false,"given":"Isidor","family":"Minovi\u0107","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8847-2704","authenticated-orcid":false,"given":"Saskia K.","family":"Klein","sequence":"additional","affiliation":[{"name":"Department of Hematology, University Medical Center Groningen, University of Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hanneke N. G.","family":"Oude Elberink","sequence":"additional","affiliation":[{"name":"Department of Allergology, University Medical Center Groningen, University of Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands"},{"name":"Groningen Research Institute for Asthma and COPD (GRIAC), University Medical Center Groningen, University of Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ido P.","family":"Kema","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andr\u00e9 B.","family":"Mulder","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.jaip.2025.10.046","article-title":"Diagnosis and Management of Patients with Mast Cell Activation Syndromes: Status 2026","volume":"14","author":"Akin","year":"2026","journal-title":"J. Allergy Clin. Immunol. 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