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Updated: May 4, 2026
Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Published on: September 20, 2016
CALR frameshift mutation detection in myeloproliferative neoplasms by microfluidic chip analysis
Michael P Greenwood1, Keith M Newton1, Kristi L Pepper1
1Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX, United States.
Background:
CALR mutation analysis is routinely used to diagnose BCR/ABL1-negative myeloproliferative neoplasms. The 2 most common CALR mutations are a 52-base pair (bp) deletion and a 5-bp insertion, which account for approximately 85% of cases.
Methods:
To evaluate our new microfluidic chip assay, we tested CALR mutant and wild-type specimens that were previously analyzed using conventional methods at a reference laboratory. Samples included EDTA-anticoagulated peripheral blood and bone marrow specimens, air dried bone marrow aspirate smears, and formalin-fixed, paraffin-embedded bone marrow sections. CALR exon 9 was PCR amplified using 2 previously published primer pairs and a third unique primer pair designed for our new assay. Amplicons were sized using microfluidic chip analysis.
Results:
Concordance with the reference method was 100% (42/42). Intra-run and inter-run reproducibility were also 100% (3/3 and 3/3, respectively). The limit of detection was confirmed to be 6% mutant alleles.
Conclusion:
We determined that the microfluidic chip assay to detect CALR exon 9 mutations was acceptable for clinical use. Compared with the conventional method, the microfluidic analysis assay benefits from a streamlined workflow, faster turnaround, and a smaller instrument footprint.
