Hemoglobin Variants That Produce Abnormal Elevations In Hb A1c In The Clinical Laboratory: Electrophoretic Patterns, HPLC Analysis and Globin Gene Sequencing
Authors: Harris Neil S. , Chui David H.K. 
Published: October 2019

Introduction
There are two commonly-used methods for measuring Hemoglobin A1c (Hb A1c) – chromatographic and immunochemical. Immunochemical analysis depends on a specific antibody directed towards an epitope that consists of the linked glucose as well as the N-terminal betachain.
High-performance liquid chromatography (HPLC)-determined Hb A1c concentrations that are unusually high (above 18%) may be a consequence of a hemoglobinopathy. Standard alkaline and acid gel electrophoresis and even an extended HPLC chromatogram may not fully resolve the abnormal hemoglobin. Globin gene analysis is an invaluable aid in such a situation.
Over the course of a 12-month period, 4 individuals were identified in whom the HbA1c fraction exceeded 20%. All cases were encountered as part of our routine clinical chemistry hemoglobin A1c service. All cases were shown to be a result of heterozygosity for an uncommon or rare hemoglobin variant. This resulted in an early-eluting variant that was close to, or coincident with, the Hb A1c peak. Only one of these 4 subjects had anemia and microcytosis, apparently related to iron deficiency and chronic kidney disease.

Methods & Materials
Hb A1c quantitation was carried out using EDTA-anticoagulated blood on a Bio-Rad Variant II HPLC analyzer (Bio-Rad Laboratories, Hercules CA). Gel electrophoresis was performed on a SPIFE 3000 system (Helena Laboratories, Beaumont TX) using spun, washed red cell pellets. Extended-run HPLC analysis utilized a BioRad D-10™ Hemoglobin Analyzer in the A2/F mode. Globin gene analysis was carried out at Boston University Medical Center using the residual washed whole blood pellet. The globin genes were amplified by PCR, the amplicons were purified, and nucleotide sequencing was performed.
Case Descriptions
Case 1. Female 58 y. Hb A1c23.8%.
o Hb 13.6 g/dL, Hct 41.2%, MCV 94 fL, RBC 4.39 x 106 per mm3, RDW 13.4%
o Alkaline gel electrophoresis: Very fast variant band, 23% of total Hb.
o Acid Gel electrophoresis: Normal.
o Globin gene analysis: Heterozygous for Hb I (α2 codon 16 AAG>GAG or Lys16Glu).
Case 2. Female 66 y. Hb A1c46%.
o Hb 7.3 g/dL, Hct 24.9 %, MCV 70 fL, RBC 3.5 x 106 per mm3, RDW 16.5%. Iron deficient with low serum ferritin of 8.3. The patient also had a history of diabetes and chronic kidney disease.
o Alkaline gel electrophoresis: “Fast” hemoglobin, but migrating very close to & slightly anodal of Hb A.
o Acid Gel electrophoresis: Variant hemoglobin migrates with Hb F
o D-10 HPLC A2/F Mode: 45% A1c
o Globin gene analysis: Heterozygous for Hb Hope, β chain codon 136 GGT>GAT or Gly136Asp
Case 3. Female 47 y. Hb A1c 43%.
o Hb 12.6 g/dL, Hct 41.7%, MCV 96.2 fL, RBC 4.33x 106 per mm3, RDW 22.9%
o Alkaline gel electrophoresis: Normal.
o D-10 HPLC A2/F Mode: Variant peak, 46% eluting between Hb A1c and A0
o Globin gene analysis: Heterozygous for Hb Moriguchi, β codon 97 CAC>TAC or His97Tyr.
Case 4. Female 83 y. Hb A1c 41% as “labile Hb A1c” or 51% as Hb A1c by extended run in A2/F mode.
o Hb 13.8 g/dL, Hct 43.4%, MCV 88.5 fL, RBC 4.9 x 106 per mm3, RDW 14%
o Alkaline gel electrophoresis: Normal.
o Acid Gel electrophoresis: Variant band, slightly anodal to Hb F
o D-10 HPLC A2/F Mode: 51% Hb A1c
o Globin gene analysis: Heterozygous for a novel variant hemoglobin, β codon 2 CAC>AAC or Histidine to Asparagine

Conclusions
Hb A1c analysis by HPLC frequently reveals the presence of a variant hemoglobin in asymptomatic individuals who have the S, C or E trait. In such cases, Hb A1c can be accurately determined despite the presence of the abnormal hemoglobin. We describe 4 individuals with marked elevation of Hb A1c. All these individuals were heterozygous for uncommon globin chain variants due to single point mutations. One subject had a Hb variant that had not been previously recognized. It is unclear whether the immunochemical assays might experience interference from these unusual variants.

Disclosures:
No relevant conflicts of interest to declare.