Haemoglobin J-Baltimore can be detected by HbA1c electropherogram but with underestimated HbA1c value

Glycated haemoglobin (HbA1c) is considered the gold standard for assessing diabetes compensation and treatment. In addition, fortuitous detection of haemoglobin variants during HbA1c measurement is not rare. Recently, two publications reported different conclusions on accuracy of HbA1c value using capillary electrophoresis method in presence of haemoglobin J-Baltimore (HbJ). Here we describe the fortuitous detection of unknown HbJ using capillary electrophoresis for measurement of HbA1c. A patient followed for gestational diabetes in our laboratory presented unknown haemoglobin on Capillarys 2 Flex Piercing analyser which was identified as HbJ. HbJ is not associated with haematological abnormalities. High Performance Liquid Chromatography methods are known to possibly underestimate HbA1c value in the presence of this variant. This variant and its glycated form are clearly distinguished on electropherogram but HbJ was responsible for underestimating the true area of HbA1c. Capillary electrophoresis is a good method for detecting HbJ but does not seem suitable for evaluation of HbA1C value in patients in presence of HbJ variant.


Introduction
Glycated haemoglobin (HbA 1c ) is considered the gold standard for assessing diabetes compensation and treatment. In addition, fortuitous detection of haemoglobin variants during HbA 1c measurement is not rare. Recently, an analytical evaluation of Capillarys 2 Flex Piercing (C2FP, Sebia, Lisses, France) has been published, assessing the possible interference of the most common haemoglobin variants in African immigrants to the United States (1). The authors concluded that those common African haemoglobin variants, notably heterozygous for haemoglobin S (HbS) or haemoglobin C (HbC), not only could be detected, but also do not interfere with HbA 1c results and do not impair the ability of HbA 1c to detect abnormal glucose tolerance on C2FP analyser. Although heterozygous for HbS or HbC are the most frequently encountered variants in France they are not the only ones (2). In 2013, we introduced C2FP instrument to our clinical biology laboratory at Rouen University Hospital, France to perform HbA 1c . Herein we report a case of haemoglobin J-Baltimore detected by electropherogram and discuss the consequences of the presence of this haemoglobin variant on accurate interpretation of HbA 1c result obtained by C2FP analyser.

Materials and Methods
A 28-year-old pregnant woman at 29 weeks of amenorrhoea, with no medical history, was referred by her attending physician at our Department of Endocrinology for management of newly diagnosed gestational diabetes. An oral glucose tolerance test and HbA 1c measurements were done respectively on Cobas 8000 analyser (Roche, Meylan, France) and C2FP instrument (Sebia, Lisses, France) in our clinical biology laboratory at Rouen University Hospital to monitor her diabetes. Oral glucose tolerance test was performed with 75 g of glucose dissolved in 250 mL of water which was absorbed by the patient.

Results
Fasting glucose, 1 hour and 2 hour levels after glucose charge were measured respectively as 4.8, 10.1 and 8.5 mmol/L. We detected two peaks of unknown haemoglobin on electropherogram. The higher peak was quantified at 47.7%. The other peak, probably corresponding to a glycated form of the first, was quantified at 1.2% ( Figure 1). C2FP software does not allow HbA 1c calculation because of insufficient separation between the peak of unknown haemoglobin and HbA 1c fraction. Therefore, the sample was tested by High Performance Liquid Chromatography (HPLC) Bio-Rad Variant II analyser (BioRad, Marnes-la-Coquette, France). HbA 1c was measured at 4.6% (27 mmol/mol) with the presence of an unknown peak in P3 window, the window on Variant's chromatogram where haemoglobin variant could be detected, quantified at 44.3%. The manufacturer's instructions define a cut-off in the window as 5% for detection of haemoglobin variant sample. The sample was analysed by the National Haemoglobinopathy Reference Laboratory (Créteil, France) which identified this haemoglobin as J-Baltimore beta 16(A13) Gly>Asp (HbJ). On electropherogram, we clearly observed insufficient separation of HbA 1c fraction from HbJ with an underestimation of HbA 1c fraction area determined by the C2FP software. We pursued our investigation to measure this interference. Thus, manual off-line recalculation of HbA 1c percentage was performed, excluding both unknown peaks. The formula used was %HbA 1c = [HbA 1c ] / ( [HbA 1c + HbA 0 ] ) × 100, then we applied the calibration equation of the software, and the HbA 1c value obtained was 4.1% (21 mmol/mol). The laboratory did not communicate this manual off-line recalculation of HbA 1c to Department of Endocrinology because of the underestimation of HbA 1c was not quantifiable.

Discussion
Fortuitous identification of haemoglobin variants during HbA 1c measurement is not common and requires screening expertise by the clinical pathologist (3). In our laboratory, we perform 12,000 tests for HbA 1c measurement per year and we identify approximately 30 cases of haemoglobin variant per year. HbJ was first described in 1963 by Baglioni and Weatherall in a black American family (4). Haematological abnormalities are not usually associated with this variant. The analytical impact of this rare variant is well known on HbA 1c measurement by HPLC (5,6). Only a few recent publications have been published on capillary electrophoresis (CE) methods (7,8). Barakat  Electropherogram with software alarm "atypical profile" presents two peaks of unknown haemoglobins. The higher unknown peak is haemoglobin J-Baltimore (HbJ) and the smaller unknown peak is glycated form of HbJ (HbJ 1c ). We observe usual fractions of haemoglobin: haemoglobin A (HbA 0 ), A1c (HbA 1c ), A2 (HbA 2 ). On this electropherogram, we observed insufficient separation of HbA 1c fraction from HbJ.

HbJ1c
HbA1c HbA2 HbA0 HbJ methods and subsequent risk of underestimating HbA 1c value (5,6). Rhea reported the same results between HPLC method, known with a negative bias, and CE (7). Little considered that the HbA 1c results of the two samples tested were accurate with CE method (8). Here, we clearly observe that we are able to identify the presence of all the fractions of interest of HbA, HbA 1c , HbJ and HbJ 1c on electropherogram obtained by C2FP analyser. We previously reported that manual off-line recalculation could be useful when CE software does not allow HbA 1c calculation (9).
In conclusion, in this case HbJ was responsible for underestimating the true area of HbA 1c , and con-sequently the proposed manual off-line recalculation of HbA 1c value. Although C2FP HbA 1c method can detect HbJ variant, the presence of this haemoglobin variant affects the accuracy of C2FP method for HbA 1c measurement.