Qualitative PCR Detection of NPM1 A, B & D Mutations in Acute Myeloid Leukemia (AML)

The TRUPCR® NPM1 Qualitative Kit is a sensitive, CE-IVD certified in vitro diagnostic assay developed for the qualitative detection of NPM1 gene mutations, specifically Type A (c.860_863dupTCTG), Type B (c.863_864insCATG), and Type D (c.863_864insCCTG). The assay uses real-time PCR with fluorescent probes and is based on a two-tube format that includes a mutation-specific primer-probe mix and an internal control gene. The kit is intended for use with genomic DNA extracted from peripheral blood samples and supports diagnosis, risk classification, and clinical decision-making in patients with acute myeloid leukemia (AML).

Molecular Background

Acute myeloid leukemia (AML) is a genetically diverse malignancy of the bone marrow characterized by clonal expansion of immature myeloid cells. One of the most frequently mutated genes in AML is Nucleophosmin 1 (NPM1), found in approximately 35% of all AML patients and in up to 60% of cytogenetically normal AML (CN-AML) cases.

NPM1 is a multifunctional protein located primarily in the nucleolus, playing essential roles in ribosome biogenesis, histone chaperoning, and pre-ribosomal transport. Mutations, predominantly found in exon 12, lead to a frame-shift and creation of a new nuclear export signal, resulting in aberrant cytoplasmic localization of NPM1 (NPM1c+)—a key hallmark of the disease.

Over 50 NPM1 variants have been described, but more than 95% of cases involve the same region, particularly:

• Type A (c.860_863dupTCTG) – the most common
• Type B (c.863_864insCATG)
• Type D (c.863_864insCCTG)

These mutations are considered early initiating events and play a significant role in AML pathogenesis and prognosis. Importantly, NPM1 mutations are associated with favorable outcomes, especially in the absence of FLT3-ITD, and have become a standard molecular marker in AML classification.

Assay Performance & Utility

The TRUPCR® NPM1 Qualitative Kit enables accurate and sensitive detection of the most clinically relevant NPM1 mutations in a single real-time PCR workflow. The assay supports:

• Initial diagnosis and molecular classification of AML
• Detection of low-level mutations with a sensitivity of 1% mutant allele
• Stratification of AML patients into prognostic groups
• Monitoring of residual disease in follow-up settings

The kit is optimized for use in routine diagnostic laboratories, providing fast, reproducible results and compatibility with widely used real-time PCR platforms.

Technical Specifications

The TRUPCR® NPM1 Qualitative Kit is a two-tube real-time PCR assay using fluorescent probe-based detection. It is designed to amplify regions of exon 12 of the NPM1 gene, where mutations A, B, and D occur, and includes a reference control gene for result validation.

• Sample Type: Genomic DNA from peripheral blood
• Input Requirement: 50–200 ng DNA per reaction
• Target Mutations: NPM1 A, B, and D (exon 12)
• Detection Method: TaqMan-based real-time PCR
• Sensitivity: Detects down to 1% mutant allele in a wild-type background
• Assay Format: Two-tube system (mutation + internal control)
• Time to Result: ~90 minutes
• Instrument Compatibility: ABI 7500, Bio-Rad CFX96™, Roche LightCycler®, and similar systems