Identification of Rare Hemoglobin Variants Manifesting as *Doublet* Hemoglobin A1c Chromatographic Peaks and Interfering With Hemoglobin A1c Measurement by High Performance Liquid Chromatography

Document Type

Conference Proceeding

Publication Date

10-2025

Publication Title

Clinical Chemistry

Abstract

BACKGROUND: Hemoglobin A1c (A1c) aids in the diagnosis and monitoring of diabetes. Our laboratory utilizes a rapid (< 1 minute), semiautomated high-performance liquid chromatography (HPLC) method (Bio-Rad D-100) to chemically separate and quantify Hemoglobin A1c in whole blood. This method also separates A1c from the common hemoglobin variants Hemoglobin S, C, D, E, or F. The FDA has approved the use of the D-100 A1c method if the patient is heterozygous for any of these variants. The D-100 method includes on-board software for evaluating A1c peak shape and retention times, as well as other chromatographic patterns that suggest a problem sample. Rarely (approximately 1 in 30,000 samples) the A1c peak manifested as an unresolved “doublet” flagged by the D-100 software (see figure). The cause of the interference was unclear. Possible explanations included sample contamination, sample instability, colorimetric interference, or chromatographic artifact. In each case, the “doublet” pattern prevented accurate quantitation of A1c in these patients. No large unknown peak was present to suggest a heterozygous variant, so this seemed an unlikely explanation, but this possibility was investigated. METHODS: Five of the rare samples exhibiting a doublet A1c pattern on the D-100 were selected for further study. In-house analytical methods employed to identify hemoglobin variants included ion-exchange HPLC (Bio-Rad Variant), capillary electrophoresis (Sebia Capillarys), and alkaline/acid agarose gel electrophoresis (Sebia Hydragel). Rare variants required reference laboratory services (Mayo Clinic Laboratories) to fully identify hemoglobin variants by isoelectric focusing and mass spectrometry. Study of these remnant samples was approved (Corewell lRB #2025-011). RESULTS: Among the five samples, four rare hemoglobin (Hb) variants were identified: Hb Hofu, Hb Tyne, Hb Athens-GA, and Hb Tacoma (in two of the samples). Each variant was readily detected by HPLC, except Hb Tacoma, which showed only a slight increase in a small “Unknown” peak at retention time 1.20 minutes. Capillary electrophoresis detected each variant except Hb Tyne, which co-eluted with Hb A. Tyne was also undetectable in alkaline and acid gel electrophoresis. Reference lab findings indicated the heterozygous variants were present at 37-51% of total hemoglobin, depending on the variant. CONCLUSION: The D-100 HPLC method rapidly and effectively separates A1c from most common hemoglobins, yet A1c “doublets” can be observed in rare samples with no other indication that an interfering variant is present. One likely explanation is that the rare hemoglobin variants are themselves glycated and partially resolved from A1c by this method. The on-board proprietary algorithm used to calculate A1c cannot account for the additional glycated hemoglobin variant, but does flag the sample for review. Our findings are important for other D-100 users and for the ongoing diabetes care of patients with such rare hemoglobin variants. Hemoglobinopathy evaluation may be uninformative if variants such as Hb Tacoma or Hb Tyne are missed on initial Hb variant screening. The impact of such rare variants is not well understood for many chromatographic, immunological, or enzymatic A1c assays. Other assays to monitor glycemic control such as fructosamine or glycated albumin should be considered.

Volume

71

Issue

Suppl1

First Page

i196

Comments

ADLM (Association for Diagnostics and Laboratory Medicine) 2025 Annual Meeting and Clinical Lab Expo, July 27-31, 2025, Chicago, IL

Last Page

i196

DOI

10.1093/clinchem/hvaf086.464

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