High-sensitivity KIT D816V variation analysis by droplet digital polymerase chain reaction: The reference laboratory perspective.

Objective: Systemic mastocytosis is a hematologic malignancy characterized by clonal expansion of neoplastic mast cells. Detection of this variation is critical for screening and diagnosis, with recent guidelines emphasizing the need for high-sensitivity assays that identify variants at a variant allele frequency below 0.05%. Our reference laboratory offers droplet digital polymerase chain reaction (ddPCR) for detection of KIT D816V at a limit of detection of 0.03% variant allele frequency-substantially higher sensitivity than next-generation sequencing (NGS).

Methods: Because high-sensitivity KIT D816V testing is still not widely available, we present our 3-year experience with KIT D816V ddPCR in a clinical setting. From January 2021 to March 2024, KIT D816V variation was detected in 14.9% (1232/8272) of samples.

Results: Peripheral blood and bone marrow positivity rates were 11.1% and 34.9%, respectively. Among 181 samples tested by both ddPCR and NGS, ddPCR identified 37.6% as positive, while NGS identified only 6.0% as positive. Next-generation sequencing showed 16% sensitivity and 100% specificity for KIT D816V detection compared with ddPCR as the gold standard, which detected the variant in 84% more samples because of its lower limit of detection. A 20-ng/mL serum tryptase threshold to screen for detecting KIT D816V by ddPCR had 73.7% sensitivity and 91.2% specificity, but lowering the serum tryptase threshold to 11.5 ng/mL increased sensitivity to 97.5%, with 70.7% specificity.

Conclusions: Overall, ddPCR for detection of KIT D816V dramatically increases sensitivity over NGS tests used for myeloid malignancies, including systemic mastocytosis. Our findings also provide support for the use of a lower serum tryptase threshold (>11.4 ng/mL instead of >20ng/mL) to initiate workup for a mast cell neoplasm.