A high stable sample loading for analysis of adult alpha-thalassemia via the improved microarray isoelectric focusing of Hb species

The isoelectric focusing has realized various improvements, including the protocols and creation of mIEF (microcolumn isoelectric focusing) instruments with excellent sensitivity for screening of diabetes and beta thalassemia. However, the problem of manual sample loading and hydration for the mIEF limits the operational capacity for stably detecting and quantitating most abnormal hemoglobin (Hb). Herein, we provided a high stable sample loading protocol for analysis of alpha thalassemia and Hb variants. In contrast to the previous volume of 20 μl, a 100 µl blood sample solution in this protocol was optimized with mixture of 6.4–7.5 and 3–10 pH carrier ampholytes, pI markers and loaded for 30 mins IPG microcolumn hydration. The hydrated microcolumn was then automatically loaded onto the mIEF chip array to which CH₃COOH and NH₄OH act as anodic and cathodic solutions. Lastly, the IEF was run for 9 mins. Hb H, Barts, A_1c, F, A₂ and CS were simultaneously separated and focused with higher resolution and sensitivity in quantifying H and Barts as low as 0.6 and 0.5 % respectively. Accordingly, there was an enhanced stability and linearity with a rapid assay time of 45 secs per sample. Moreover, analysis showed a fitting linear relationship with conventional technology at R² = 0.9803 for H and R² = 0.9728 for Barts thereby indicating greater accuracy confirmed by the AUC. Hence, the developed protocol could simply be employed for high stable and throughput batch sample loading of hydration, and accurate separation and quantitation of Hb variants for alpha and beta thalassemia.