title: Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC.
abstract: OBJECTIVE
  To investigate the underlying cause of unexpectedly low HbA1c results (3.3-3.5%) obtained by HPLC in three siblings undergoing routine screening for type 2 diabetes mellitus.
  
  
  METHODS
  HbA1c was measured using an alternate method based on a different analytical principle (the Siemens DCA 2000+ immunoassay). Hemoglobin fractionation was performed by HPLC on the BioRad Variant II, gel electrophoresis at acid and alkaline pH on the Sebia Hydrasys 2, and capillary electrophoresis on the Sebia Capillarys 2. Sequencing of the beta globin gene was also conducted.
  
  
  RESULTS
  HbA1c analysis by immunoassay gave significantly higher results, ranging from 5.2-5.5%. Hemoglobin fractionation by HPLC showed an abnormal peak comprising approximately 43% of total hemoglobin, suggesting the presence of a beta chain hemoglobin variant. Gel electrophoresis at alkaline pH revealed a very unusual pattern, with 3 abnormal bands migrating with Hb F, between Hb F and Hb S, and slightly cathodal to Hb S. A single band in the Hb A position was seen on gel electrophoresis at acid pH. Capillary electrophoresis revealed two abnormal peaks, comprising 42% and 5% of total hemoglobin. Sequencing of the beta globin gene showed heterozygosity for Hb Hirose (beta 37(C3) Trp>Ser), an extremely rare variant with a substitution at the α1β2 interface.
  
  
  CONCLUSIONS
  We describe the chromatographic and electrophoretic properties of Hb Hirose, and demonstrate that this rare variant causes falsely low HbA1c results on the BioRad variant II Turbo 2.0. Recognition of this interference is crucial in order to prevent reporting erroneous results.
authors.affiliation: DynaLIFE(Dx), Edmonton, Canada; Department of Laboratory Medicine and Pathology, University of Alberta, Canada. Electronic address: mathew.estey@dynalifedx.com.
authors.forename: Mathew P
authors.initials: MP
authors.lastname: Estey
authors.affiliation: DynaLIFE(Dx), Edmonton, Canada; Department of Laboratory Medicine and Pathology, University of Alberta, Canada.
authors.forename: Karina
authors.initials: K
authors.lastname: Rodriguez-Capote
authors.affiliation: Synergy Medical Clinic, Sherwood Park, Canada.
authors.forename: Tolulope
authors.initials: T
authors.lastname: Adelowokan
authors.affiliation: DynaLIFE(Dx), Edmonton, Canada.
authors.forename: Trefor
authors.initials: T
authors.lastname: Higgins
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary; D005189:False Positive Reactions; D006442:Glycated Hemoglobin / Q000378:metabolism*; D006455:Hemoglobins, Abnormal / Q000032:analysis*; D006801:Humans
chemical_list: D006442:Glycated Hemoglobin; D006455:Hemoglobins, Abnormal; C517652:hemoglobin A1c protein, human; C040845:hemoglobin Hirose
keywords: Electrophoresis; HbA1c; Hemoglobin Hirose; Hemoglobinopathy; High performance liquid chromatography