The Efficiency of Capillarys 2 Flex Piercing, Premier Resolution-HPLC, and VARIANT II-HPLC for Hemoglobin Constant Spring Detection

Hemoglobin Constant Spring (HbCS) is the most common non-deletional α-thalassemia mutation in Southeast Asia (SEA) and is frequently missed during routine Hb analysis because of its extremely low expression in peripheral blood. This study aims to evaluate the efficacy of three hemoglobin analysis systems: Capillarys 2 Flex Piercing (CE), Premier Resolution (PR)-HPLC, and VARIANT II-HPLC for the detection of HbCS. A total of 40 EDTA blood samples suspected of carrying HbCS were analyzed. Hb analysis was performed using the three analytical systems, while molecular confirmation of Hb CS/Paksé (PS) mutations was carried out using multiplex AS-PCR. In addition, α0-thalassemia deletions (–SEA, –THAI, and –CR) were identified using real-time PCR with high-resolution melting analysis. Four Hb typing patterns were identified: CSA2A (n = 26), CSA2ABart’sH (n = 8), CSEABart’s (n = 4), and CSEA (n = 2). Molecular analysis confirmed the presence of the HbCS mutation in all 40 samples. The correct identification rates were 90.0% for CE and 87.5% for both PR-HPLC and VARIANT II-HPLC. Misidentification occurred most frequently in the CSA2A group, likely due to the extremely low proportion of HbCS in individuals without co-inherited α-thalassemia. In addition, PR-HPLC misidentified HbCS in one sample from each of the CSA2ABart’sH, CSEABart’s, and CSEA groups. These findings suggest that CE provides higher efficiency than HPLC systems for detection of HbCS. However, molecular confirmation remains essential for accurate diagnosis.