Interference of Hemoglobin (Hb) Hope on Measurement of HbA1c Using an HPLC Method

Hemoglobin A1c (HbA1c) can be measured using different testing modalities and, depending on the method applied, several factors may interfere in its measurement, which may lead to falsely abnormal results. ¹

We report on 2 non-related patients from Mali with suspected hemoglobinopathy. Blood samples from both patients were processed in the HPLC Bio-Rad D-10^TM (D-10) Dual Program analyzer (Bio-Rad Laboratories Inc, Hercules, CA), the chromatograms from both patients revealed an abnormal pattern. Showing a high peak of the HbA1c and abnormally high values of HbA1c.

The 2 samples were processed by another method for the HbA1c measurement, the Immunoturbidimetric Tina-quant® HbA1c Gen.3 assay, in a Cobas® 8000 (Roche Diagnostics, Rotkreuz, Switzerland). The measurements, taken in accordance with IFCC standardization, revealed HbA1c values that were consistent with the FPG concentrations obtained in both patients (Table 1).

Table 1.

Blood Test Levels Corresponding to Patients (A and B) with the Hb Hope Variant.

Patient	Age (yr)	Sex	FPG (mg/dL)	HbA1c Bio-Rad D-10TM (mmol/mol (%))	HbA1c Cobas (mmol/mol (%))	Hb Total (g/dL)	DNA sequencing
A	16	m	92	927 (87.0)	29 (4.8)	13.9	Hb Hope and Hb S
B	23	f	108	272 (27.4)	31 (5.0)	8.1	Hb Hope and Alfa-thalassemia

FPG, fasting plasma glucose; HbA1c, hemoglobin A1c; HPLC Bio-Rad D-10^TM Dual Program analyzer; Cobas, Tina-quant HbA1c Gen 3 in a Cobas 8000 of Roche Diagnostics.

In cation exchange D-10, an abnormally high level of HbA1c (Table 1) has been noted. Since such a high level of HbA1c is incompatible with physiological possibilities, we suspected the presence of a hemoglobin variant co-eluated with HbA1c. In order to identify this variant, we performed the molecular study of β-globin gene and DNA sequencing.

Sequencing the Hb β-chain gene from each patient revealed heterozygosity for the Hb Hope [β136 (H14) Gly→Asp (GGT→GAT)], patient-A presents Hb Hope in association with heterozygosity for the Hb S[β6 (A3) Glu→ Val (GAG→ GTG)]; and patient-B presents Hb Hope in association with heterozygosity for Alfa-thalassemia.

Hb Hope is a clinically asymptomatic unstable variant of the β-globin chain that shows a lower oxygen affinity thus explaining its usual innocuity. ² Hb Hope was reported in several African-American, Japanese, Thais, Laotians, Mauritanian, Cubans, and Spanish families, ³ but the origin of this variant still remains unanswered.

Hb Hope is characterized by a comparable charge altering mutation (β136 Gly→Asp) and the intra chain salt bridge so formed between the carboxyl group of β136 Asp and the charged α-amino group of β1 valine. ⁴ This salt bridge neutralizes the positive charges to the extent that HbA1c and Hb Hope co-elute from the HPLC column. So, in our HPLC method, Hb Hope and HbA1c elute in the same window leading to probable overestimation, like in other studies.^5,6

It is possible that alpha-thalassemia in patient-B justifies the lower overestimation of HbA1c in relation to patient-A in D-10 (Table 1).

We conclude that, in diabetes patients who are heterozygous for the Hb Hope variant, determining HbA1c levels using the D-10 HPLC method gave falsely high results due to an abnormal separation of HbA1c. We, therefore, recommend that all chromatograms be thoroughly reviewed prior to the release of a HbA1c result to identify possible interference from Hb variants, particularly when the result is inconsistent with the patient’s fasting blood glucose concentrations or medical history. Additionally, in Hb Hope variant carriers, we recommend measurement of HbA1c using other alternatives, such as a turbidimetric immunoassay Tina-quant HbA1c Gen.3, which is less likely to be subject to interference when the mutations are present much farther from the N-terminus of the Hb β-chain.