Clinica Chimica Acta 465 (2017) 80–81

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Clinica Chimica Acta

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Letter to the Editor

Hemoglobin J-Iran (HBB c.232CN G): of HbA1c measurement in CE-HPLC method. HbA1c level was measured
Interference with the assay of HbA1c by immunoturbidimetric assay in Dimension RxL Max instrument (Sie-
mens, Malvern, PA). HbA1c level was reported as 6.5%. DNA mutation
analysis was performed to detect the abnormal hemoglobin variant in
Keywords:
Hb J-Iran (HBB c.232CNG) the department of genetics, Antalya Education and Research Hospital.
HbA1c DNA isolation was achieved by the salt precipitation method. The 3
Cation-exchange HPLC exons coding for the β globin gene as well as the promoter, first intron,
5′UTR and 3′UTR sequences were replicated by polymerase chain reac-
tion (PCR) using suitable primers. This was followed by automated DNA
sequencing for mutation analysis. Presence of heterozygote Hb J-Iran
Glycated hemoglobin (HbA1c) is the gold standard for the assess- variant (HBB c.232C N G) was detected in the patient.
ment of long-range glycemic control in patients with diabetes [1]. CE-HPLC separates Hb species based on charge differences between
Methods of HbA1c analysis can be divided into two categories: methods HbA1c and other hemoglobins. If the Hb substitution causes a change in
based on molecular charge and those based on structure. The former the net charge of the Hb (as with Hb variants S, C, D, and E), then it may
category includes cation-exchange HPLC (CE-HPLC) and electrophore-
sis, and the latter includes immunoassays, boronate affinity chromatog-
raphy, and mass spectrometry [2]. Although CE-HPLC is widely used for
the measurement of HbA1c, presence of hemoglobin variants may inter-
fere this measurement, producing falsely high or low values when the
hemoglobin variant or its glycated form cannot be separated from he-
moglobin A or HbA1c [3]. The Hemoglobin J-Iran (HBB c.232CN G) vari-
ant, first reported in Iran, was subsequently found in Turkey and in a
Russian-Armenian family [4,5]. The mutation in codon 77 (CAC N GAC)
which caused the β77 His → Asp substitution in the β chain is present
in Hb J-Iran. The substitution at β77 leads to a higher negative charge
on the surface of the βJ-Iran subunit, which enhances its electrostatic at-
traction for the normal positively charged α subunit. Patients with Hb J-
Iran have no apparent health problems related to the hemoglobin vari-
ant and do not need blood transfusion [4]. The interference of Hb J-Iran
variant on the measurement of HbA1c is not recorded in the HbVar da-
tabase [6]. Herein we report a case of Hb J-Iran variant that interferes the
measurement of HbA1c by CE-HPLC method.
A forty-four years old female patient with suspicion of type 2 diabe-
tes mellitus had no complaints related with the presence of hemoglo-
binopathy. Complete blood count (CBC) and fasting glucose
measurements were done in Coulter LH 780 Analyzer (Beckman Coul-
ter, Holliston, MA) and in AU5800 Analyzer (Beckman Coulter,
Holliston, MA), respectively. HbA1c was measured by CE-HPLC method
in HLC-723 G7 Analyzer (Tosoh Bioscience, San Francisco, CA). CBC was
as follows: RBC: 4.3·1012/L, Hb: 12.7 g/dL, Hct: 37.1%, MCV: 85.6 f/L.
Fasting glucose was 102 mg/dL. An unusual peak of 45.1% was observed
in the labile HbA1c window (retention time of 0.62 min) and stabile
HbA1c was 0.0% (Fig. 1). The HbA1c level could not be reported. Pres-
ence of such a high level of labile HbA1c alerted us for a possible pres-
ence of hemoglobinopathy. Then the patient was advised to have a
hemoglobin variant analysis. The HbA, HbA2 and HbF levels were also
analyzed by HPLC using the short program for the Variant II (Bio Rad,
Hercules, CA). CE-HPLC revealed HbA: 45.1%, HbA2: 2.3%, and HbF:
1.3%. A peak of 32.5% unknown hemoglobin was detected at 1.22 min
of hemoglobin chromatogram. According to these results we found Fig. 1. Chromatogram of HbA1c analysis with the CE-HPLC (Tosoh HLC-723 G7) method.
that unknown hemoglobin variant was responsible for the interference The unusual peak was labeled in the LA1c window.

http://dx.doi.org/10.1016/j.cca.2016.12.015
0009-8981/© 2016 Elsevier B.V. All rights reserved.
 Letter to the Editor
cause interference with methods such as CE-HPLC or electrophoresis
[7]. HPLC-based ion-exchange methods for HbA1c have recently been
automated, and interferences by Hb species such as HbF and HbS have
been minimized. However, several reports have described artificially
low or high HbA1c results with hemoglobin variants such as Hb
Wayne, Hb Haelen and others when an automated CE-HPLC method is
used [8,9]. Hb J-Iran is one of the hemoglobin variants that initially
have been discovered in Iran. There is a little knowledge about the com-
plications caused by Hb J-Iran [10]. Our patient was recognized to have a
high level of labile HbA1c causing interference in CE-HPLC method. We
think that β77 His → Asp substitution is responsible for this interfer-
ence. Because the substitution at β77 leads to a higher negative charge
on the surface of the βJ-Iran subunit [4], Hb J-Iran and HbA1c are placed
in the same retention time in the chromatogram leading to interference.
We could not find any study about the effect of Hb J-Iran variant on the
measurement of HbA1c in the literature. Hb J-Iran variant is rarely seen
in Turkey [5]. Although the presence of Hb J-Iran does not lead to clinical
abnormalities, it also does not affect hematologic parameters. We think
that Hb J-Iran variants cases are mostly missed. Hb J-Iran variant should
be taken into account for cases that their HbA1c levels cannot be evalu-
ated by CE-HPLC method. If the CE-HPLC method is used, then careful
inspection of chromatograms may identify the presence of aberrant
peaks produced by variants. When the CE-HPLC method showed the
presence of an uncommon hemoglobin variant, an alternative method
to measure HbA1c should be used.
References
[1] J. Nadelson, S.K. Satapathy, S. Nair, Glycated hemoglobin levels in patients with de-
compensated cirrhosis, Int. J. Endocrinol. 2016 (2016) 8390210.
[2] J.M. Rhea, R. Molinaro, Pathology consultation on HbA(1c) methods and interfer-
ences, Am. J. Clin. Pathol. 141 (2014) 5–16.
[3] K. Bouzid, H.B. Ahmed, E. Kalai, et al., Prevalence of hemoglobin variants in a diabetic
population at high risk of hemoglobinopathies and optimization of HbA1c monitor-
ing by incorporating HPLC in the laboratory workup, Libyan J. Med. 27 (2014)
http://dx.doi.org/10.3402/ljm.v9.25768.
81
[4] S.J. Dehghani, A. Amiri Dashtarzhen, S. Nasirabadi, et al., Combined α-thalassemia
and Hemoglobin J-Iran (β77 His →Asp). A family study in southern Iran, Iran. Red
Crescent Med. J. 13 (2011) 586–589.
[5] A. Köseler, A. Atalay, H. Koyuncu, et al., Molecular identification of a rare hemoglobin
variant, Hb J-Iran [beta77(EF1)His → Asp], in Denizli province of Turkey, Turk. J.
Haematol. 23 (2006) 164–166.
[6] B. Giardine, J. Borg, E. Viennas, et al., Updates of the HbVar database of human he-
moglobin variants and thalassemia mutations, Nucleic Acids Res. 42 (2014)
1063–1069.
[7] R.R. Little, W.L. Roberts, A review of variant hemoglobins interfering with hemoglo-
bin A1c measurement, J. Diabetes. Sci. Technol. 3 (2009) 446–451.
[8] K. Rodríguez-Capote, M.P. Estey, V.E. Barakauskas, et al., Identification of Hb Wayne
and its effects on HbA1c measurement by 5 methods, Clin. Biochem. 48 (2015)
1144–1150.
[9] M. Bots, A.K. Stroobants, B. Delzenne, et al., Two novel haemoglobin variants that af-
fect haemoglobin A1c measurement by ion-exchange chromatography, Clin. Chem.
Lab. Med. 53 (2015) 1465–1471.
[10] S. Rahbar, D. Beale, W.A. Isaacs, H. Lehmann, Abnormal haemoglobins in Iran. Obser-
vation of a new variant—haemoglobin J Iran (alpha-2-beta-2 77 His–Asp), Br. Med. J.
1 (1967) 674–677.
Ayşegül Uğur Kurtoğlu⁎
Esin Eren
Department of Biochemistry, Antalya Education and Research Hospital,
Antalya, Turkey
⁎Corresponding author at: Varlık Mah, Kazım Karabekir cad,
Antalya Education and Research Hospital,
Department of Biochemistry, 07050 Antalya, Turkey.
E-mail address: ugurkurtoglu@yahoo.com (A.U. Kurtoğlu).
Özgür Erkal
Department of Genetics, Antalya Education and Research Hospital, Antalya,
Turkey
Erdal Kurtoğlu
Necat Yilmaz
Department of Hematology, Antalya Education and Research Hospital,
Antalya, Turkey
11 December 2016