Thalassemia diagnosis relies on precise genetic testing, which requires reliable quality control (QC) materials. Current options, such as limited clinical samples or synthetic DNA, lack both renewable supply and the ability to authentically mimic patient samples across the entire testing process. To address this gap, we developed and validated a novel panel of QC materials based on patient-derived Immortalized B-Lymphoblastoid cell lines for thalassemia genotyping. PBMCs from 12 patients with thalassemia were immortalized using an optimized EBV-transformation protocol supplemented with specific growth factors and regulators. The resulting cell lines were then prepared as QC materials by cryopreservation at a defined optimal density in a serum-free solution. We assessed the stability of these QC materials under various conditions, including freeze-thaw cycles, long-term and short-term storage, by analyzing DNA integrity via agarose gel electrophoresis, quantifying ACTB copy number using droplet digital PCR and validating reference mutant loci using gap-PCR or Sanger sequencing. Furthermore, their commutability was confirmed across five different genotyping platforms. The optimized EBV-transformation protocol significantly outperformed the basic method, increasing the transformation efficiency to 90.0% (from 53.1%, p < 0.05) and reducing the required time to 18.8 days (from 28.8 days, p < 0.05). Following resuscitation, the cryopreserved cell lines maintained stable morphology and proliferative capacity for at least 21 days in culture. Comprehensive validation confirmed the QC materials' stability, demonstrated by intact genomic DNA fragment, stable reference loci and consistent reference genes copy numbers, across various storage conditions, as well as their commutability, yielding uniform genotyping results across multiple detection platforms. We have established a comprehensive and stable QC panel for thalassemia genotyping that serves as a standardized resource for quality assurance, with the potential to be extended as a model for other genetic disorders. • Provides renewable patient-derived cell line QC panel mimics clinical samples, overcomes limits of finite & synthetic DNA. • Optimizes EBV-induced cell immortalization, enhancing efficiency and speed for large-scale QC material generation. • Offers a validated commutable resource with stable consistent multi-platform performance for lab standardization.
Zhang et al. (Mon,) studied this question.