A patient has an abnormal hemoglobin band that migrates with Hb S on cellulose acetate (pH 8.4) hemoglobin electrophoresis. The solubility test is negative. What test should be performed next?

A patient has an abnormal hemoglobin band that migrates with Hb S on cellulose acetate (pH 8.4) hemoglobin electrophoresis. The solubility test is negative. What test should be performed next?

• A. Citrate agar (pH 6.2) electrophoresis
• B. HbA2 quantitation
• C. Acid elution stain
• D. Blood film evaluation

Answer: (A)

When an abnormal hemoglobin behaves like Hb S on alkaline electrophoresis but the solubility test is negative, it suggests a different hemoglobin variant that co-migrates with Hb S at alkaline pH. To tell which variant it actually is, you use an electrophoresis method that separates hemoglobins at an acidic pH. Citrate agar electrophoresis at pH about 6.2 resolves Hb variants more clearly than alkaline gels, so HbS will separate from others such as HbD or HbG. This allows precise identification of the actual variant causing the band. HbA2 quantitation wouldn’t identify a co-migrating Hb variant or distinguish Hb S from other Hb-like variants. An acid elution stain and routine blood film evaluation can provide helpful clues about the presence of abnormal hemoglobins or sickling morphology, but they won’t definitively separate or identify the variant. Therefore, citrate agar electrophoresis is the appropriate next step.

When an abnormal hemoglobin behaves like Hb S on alkaline electrophoresis but the solubility test is negative, it suggests a different hemoglobin variant that co-migrates with Hb S at alkaline pH. To tell which variant it actually is, you use an electrophoresis method that separates hemoglobins at an acidic pH. Citrate agar electrophoresis at pH about 6.2 resolves Hb variants more clearly than alkaline gels, so HbS will separate from others such as HbD or HbG. This allows precise identification of the actual variant causing the band.

HbA2 quantitation wouldn’t identify a co-migrating Hb variant or distinguish Hb S from other Hb-like variants. An acid elution stain and routine blood film evaluation can provide helpful clues about the presence of abnormal hemoglobins or sickling morphology, but they won’t definitively separate or identify the variant. Therefore, citrate agar electrophoresis is the appropriate next step.