Biomedicine Fragmentation NGS 29 de July de 2025

Clinical Implementation of the Molecular Algorithm for Ph-Negative MPN in Collaboration with the Hospital of Jerez

From the Support and Applications Department at BIOMOL, we share a recent case of implementing a complete diagnostic workflow for Philadelphia-negative Myeloproliferative Neoplasms (Ph-negative MPNs), developed in collaboration with the Hematology Department of the University Hospital of Jerez de la Frontera.

The objective was to structure a sequential algorithm that enables accurate identification of mutations in BCR-ABL1, JAK2, CALR, and MPL, integrating technologies such as real-time PCR, HRM, and next-generation sequencing (NGS), while ensuring standardization across 7500 Fast and SeqStudio platforms.

Standardization: Equipment and Protocols

1. BCR-ABL1 – Screening and Quantification

For BCR-ABL1 screening and quantification, a transition was made from the LightCycler (LC) system to the 7500 Fast Real-Time PCR system (Applied Biosystems).
This migration was carried out without requiring additional modifications, maintaining the previous detection standards.
A specific standard curve for the p190 isoform was developed, enabling robust quantification.
A curve for the p210 isoform was also validated, although it continues to be commonly quantified using an alternative methodology, as per the laboratory’s decision.

2. JAK2 V617F

The diagnosis of this mutation, present in PV, ET, and PMF, continues to be performed using client-specific probes and primers already optimized for the 7500 system. No intervention from BIOMOL was required.

3. JAK2 Exon 12 – HRM

The protocol for High-Resolution Melting (HRM) analysis was reinstalled on the 7500 platform, particularly useful in suspected Polycythemia Vera cases with negative JAK2 V617F.
This approach allows discrimination of less common variants not detected by ASO-PCR.

4. CALR – Fragment Analysis

The CALR assay is performed via fragment analysis on the SeqStudio system.
Active technical support was provided due to challenges with visualization and analysis. After adjusting electrophoresis parameters and fragment size, interpretable and reproducible profiles were achieved.

Clinical Results and Validation

The algorithm was applied to 414 patients with suspected Ph-negative MPN:

BCR-ABL1 was negative in all cases.
40 patients were identified as JAK2 V617F positive.
For JAK2-negative patients, Exon 12 was analyzed, followed by CALR and MPL sequencing.
In a sub-cohort negative for both BCR-ABL1 and JAK2, NGS was applied to optimize the detection of non-canonical mutations.
All positive results for JAK2, CALR, or MPL were consistent with the final clinical diagnosis, supporting the robustness of the proposed algorithm.

Conclusions

The collaboration with the University Hospital of Jerez highlights the importance of specialized technical support in the implementation of complex diagnostic workflows in clinical settings.

BIOMOL’s contribution was crucial in the standardization of protocols, adaptation of platforms (7500 Fast and SeqStudio), and integration of advanced methodologies such as HRM and NGS. This structured approach ensured analytical traceability and strengthened the reliability of molecular diagnostics in patients with suspected myeloproliferative neoplasms.

This case reaffirms the value of a well-designed implementation aligned with laboratory practices, and reinforces our commitment to a more agile, traceable, and effective precision medicine.