Hemoglobin A1c (HbA1c) is routinely used to diagnose diabetes and monitor treatment. Therefore, it is important to identify factors that interfere with the accuracy of HbA1c measurement. Hemoglobin (Hb) Wayne is a frameshift alpha chain variant that produces two isoforms termed Hb Wayne I and II (1). Although clinically silent, this variant can interfere with HbA1c measurement with high performance liquid chromatography (HPLC) methods. Hb Wayne isoforms co-elute with HbF and HbA1c peaks, leading to a falsely elevated HbA1c. A few cases have been reported in the literature that inaccurate HbA1c measurement due to presence of Hb Wayne resulted in diabetes misdiagnosis (ie., HbA1c results > 6.5%) and treatment (2, 3).

Since correct identification of Hb Wayne interference is crucial when HbA1c is measured via HPLC, then the question is how to effectively detect it? A previously published study implemented an algorithm for detecting Hb Wayne interference in which samples with HbF and HbA1c co-elevations were flagged (4). However, there are challenges with sending the suspected specimen to the hematology laboratory, namely prolonged turnaround time.

With the increasing availability of mass spectrometers in the clinical laboratory and advancement of technology and data analysis software, both top-down and bottom-up Mass Spectrometry (MS) approaches have been developed and implemented for Hb variant identification. Compared to traditional methods like capillary electrophoresis, HPLC, and gel electrophoresis that all present problems of Hb variant co-migration/low resolving power; MS method provides the improved sensitivity and specificity, and most importantly one single test can detect all Hb variants without secondary analysis. Moreover, targeted molecular tests for Hb variant identification are expensive and time-consuming and could not detect certain variants. In general, top-down protein MS analysis which ionize the whole protein/Hb subunits have been more widely more adopted in by clinical labs because the sample preparation procedure is simple and rapid; diluted whole blood is directly injected into the MS without any protein digestion or pretreatment, and there is a short analytical time usually within a few minutes. After mass deconvolution by MS software, Hb variants are characterized by differences in mass shift of the Hb subunits or signature MS spectra, then the detected Hb variant is confirmed through a database search.

A new algorithm for identifying Hb Wayne interference on HbA1c HPLC would be that if cases with HbF and HbA1c co-elevation are detected, they are then investigated for concordance with glucose and HbA1c result from other platforms. Suspected cases are then analyzed by an intact MS method to identify the presence of the Hb Wayne spectra (manuscript in preparation). If Hb Wayne interference is confirmed, then a non-HPLC methodology would be needed for future HbA1c determination. By incorporating the MS Hb variant identification into the workflow, the hemoglobinopathy investigation workup is greatly shortened to within one hour. Similar algorithms can be adopted by laboratories to detect interference from other Hb variants in HPLC systems used for HbA1c measurement.

References

  1. Seid-Akhavan M, Winter WP, Abramson RK, Rucknagel DL. Hemoglobin Wayne: a frameshift mutation detected in human hemoglobin alpha chains. Proc Natl Acad Sci U S A 1976 Mar;73 3:882-6. Epub 1976/03/01 as doi: 10.1073/pnas.73.3.882.
  2. Chen J, Diesburg-Stanwood A, Bodor G, Rasouli N. Led Astray by Hemoglobin A1c: A Case of Misdiagnosis of Diabetes by Falsely Elevated Hemoglobin A1c. J Investig Med High Impact Case Rep 2016 Jan-Mar;4 1:2324709616628549. Epub 2016/02/06 as doi: 10.1177/2324709616628549.
  3. Milhem M, July M, Elhamdani S, BenHamed N. Hemoglobin Wayne Variant Interfering with Hemoglobin A1c Measurement. AACE Clin Case Rep 2019 Jan-Feb;5 1:e59-e61. Epub 2020/01/23 as doi: 10.4158/ACCR-2018-0017.
  4. Rodriguez-Capote K, Estey MP, Barakauskas VE, Burton T, Holmes D, Krause R, Higgins TN. Identification of Hb Wayne and its effects on HbA1c measurement by 5 methods. Clin Biochem 2015 Nov;48 16-17:1144-50. Epub 2015/08/08 as doi: 10.1016/j.clinbiochem.2015.07.100.