Rapid and Cost-Effective ABO Blood Genotyping Using a Freeze-Dried, Point-of-Care Ready Loop-Mediated Isothermal Amplification (LAMP) Assay.

Background: The accurate and rapid genotyping of ABO (chromosome 9q34.2) blood types is critical for clinical diagnostics and transfusion medicine, particularly in scenarios where serological methods yield uncertain results, such as in neonatal testing or with rare ABO subtypes. Methods: This study describes a loop-mediated isothermal amplification (LAMP)-based method for ABO genotyping that offers a faster and more cost-effective alternative to conventional PCR-based techniques. Results: The method targets four key single nucleotide polymorphisms (SNPs) at positions 261, 297, 703, and 930, allowing for the differentiation of common A, B, and O blood types, as well as the rare AB subtype B(A)01. The detection of the B(A)01 subtype is clinically important for preventing transfusion mismatches where serology may be inconclusive. Operating at a constant temperature, the assay can be completed in under an hour without the need for a thermocycler, offering significant time and cost benefits over qPCR. The method demonstrated high specificity, demonstrating detection down to 10 copies across all assays. When validated against a gold-standard method on clinical blood samples, the LAMP assay showed high accuracy (95% C value calculated via binomial exact method): 97.4% for type O, 98.7% for type A, 98.7% for type B, and 100% for the B(A)01 subtype. To enhance usability for point-of-care applications, freeze-dried reagents were developed that permit direct loading of lysed blood samples while maintaining high performance. Conclusions: This simplified and robust format positions the LAMP assay as a promising tool for rapid and reliable ABO genotyping in diverse clinical settings.