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Hemoglobin (Hb) A2' is a hematologically silent variant of HbA2 that is detected easily by high-performance liquid chromatography (HPLC), where it elutes in the S window. Our purposes were to define diagnostic criteria for the HbA2' trait using the Variant II (Bio-Rad, Hercules, CA) and to determine the prevalence of HbA2' in a metropolitan patient population. All Hb screens (N = 5,862) performed during a 26-month period were reviewed for new hemoglobinopathies. We identified 57 cases of HbA2' trait, making it the fourth most prevalent Hb variant detected in this population after HbS, HbC, and β-thalassemia minor. For HbA2' trait cases, the mean HbA2 level was 1.7% (SD, 0.17%), and the mean HbA2' level was 1.3% (SD, 0.18%). Six possible HbA2'/β-thalassemia double heterozygotes were identified, for whom the sum of the HbA2 and HbA2' exceeded 4% of total Hb. Hb variants that might interfere with detection of HbA2' include HbS, glycosylated HbC, and HbG2. Diagnostic criteria proposed for the HbA2' trait by HPLC are HbA2 of 2% or less, S window peak of 1% to 2%, no previous diagnosis of HbS, and absence of HbG and HbC.