HPLC Retention Time as a Diagnostic Tool for Hemoglobin Variants and Hemoglobinopathies: A Study of 60000 Samples in a Clinical Diagnostic Laboratory

BACKGROUND: Previous evaluations of HPLC as a tool for detection of hemoglobin variants have done so within newborn-screening programs and/or by use of stored samples. We describe a 32-month prospective study in a clinical diagnostic laboratory in which we evaluated the imprecision of HPLC retention times and determined the retention times for hemoglobin variants seen in a multiethnic setting. METHODS: We analyzed all samples on the Bio-Rad Variant II HPLC system. For normal hemoglobin fractions and hemoglobin variants, we recorded and analyzed their retention times, their proportion of the total hemoglobin (%), and the peak characteristics. We compared the imprecision of retention time with the imprecision of retention time normalized to the retention time of hemoglobin A0 (Hb A0) and to the retention time of Hb A2. Alkaline and acid hemoglobin electrophoresis, and in certain cases globin chain electrophoresis, isoelectric focusing, and DNA analysis, were performed to document the identities of the hemoglobin variants. RESULTS: The mean (SD) imprecision (CV) of the retention time was 1.0 (0.7)% with no statistical difference compared with the imprecision for normalized retention times. Among 60,293 samples tested, we encountered 34 unique hemoglobin variants and 2 tetramers. Eighteen variants and 2 tetramers could be identified solely by retention time and 3 variants by retention time and proportion of total hemoglobin. Four variants could be identified by retention time and peak characteristics and eight variants by retention time and electrophoretic mobility. One variant (Hb New York) was missed on HPLC. Retention time on HPLC was superior to electrophoresis for the differentiation and identification of six members of the Hb J family, four members of the Hb D family, and three variants with electrophoretic mobilities identical or similar to that of Hb C. Six variants with electrophoretic mobilities identical or similar to that of Hb S could be differentiated and identified by retention time and proportion of total hemoglobin. HPLC detected two variants (Hb Ty Gard and Hb Twin Peaks) missed on electrophoresis. CONCLUSIONS: The retention time on HPLC is reliable, reproducible, and in many cases superior to conventional hemoglobin electrophoresis for the detection and identification of hemoglobin variants. Confirmatory testing by electrophoresis can be eliminated in the majority of cases by use of retention time, proportion of total hemoglobin, and peak characteristics of HPLC.