Validation of an Automated HPLC Method for Quantification of Hemoglobin S

Abstract

HPLC is a sensitive and rapid method for separating and quantifying hemoglobin (Hb) variants (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Accurate quantification of Hb S is an essential component in the discrimination of uncomplicated Hb S trait from other conditions characterized by the presence of Hb S. This is particularly important at our institution where the initial screening of new Air Force recruits for potentially disqualifying hematologic conditions is essential before the inception of basic training activities. The Bio-Rad Variant is an automated cation-exchange HPLC method technically well suited for the rapid and accurate quantification of Hb A2 and Hb F (8). Chromatography of each sample is completed in 6 min. The system also resolves and allows presumptive identification of common Hb variants (Hb S and C) (9). Confirmation of the Hb variants detected by the system must be performed by using an alternative method. We evaluated the analytical performance of the Bio-Rad Variant HPLC System in the resolution and quantification of Hb S. Reference ranges for Hb S and Hb A2 were established for individuals with uncomplicated Hb S trait. Our reference laboratory (Brooks Epidemiology Lab) receives 250 to 300 requests for Hb electrophoresis per month. Referring facilities submit an EDTA-anticoagulated whole-blood specimen and are asked to provide complete blood count (CBC) results, a stained peripheral smear, recent transfusion history, pertinent patient history, ethnic background, age, and serum ferritin results, if available. A Hb electrophoresis panel consisting of a screen for sickle Hb, quantification of Hb fractions (normally Hb A, Hb A2, and Hb F), alkaline agarose gel electrophoresis, and acid agarose gel electrophoresis (as required for confirmation of the identity of abnormal Hbs) is performed. Specimens from 319 …