- Email: [email protected]
Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC
CLB-09185; No. of pages: 4; 4C: Clinical Biochemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Short Communication
Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC Mathew P. Estey a,b,⁎, Karina Rodriguez-Capote a,b, Tolulope Adelowokan c, Trefor Higgins a a b c
DynaLIFEDx, Edmonton, Canada Department of Laboratory Medicine and Pathology, University of Alberta, Canada Synergy Medical Clinic, Sherwood Park, Canada
a r t i c l e
i n f o
Article history: Received 22 September 2015 Received in revised form 19 November 2015 Accepted 20 November 2015 Available online xxxx Keywords: Hemoglobin Hirose HbA1c High performance liquid chromatography Electrophoresis Hemoglobinopathy
a b s t r a c t Objectives: To investigate the underlying cause of unexpectedly low HbA1c results (3.3–3.5%) obtained by HPLC in three siblings undergoing routine screening for type 2 diabetes mellitus. Design and methods: HbA1c was measured using an alternate method based on a different analytical principle (the Siemens DCA 2000+ immunoassay). Hemoglobin fractionation was performed by HPLC on the BioRad Variant II, gel electrophoresis at acid and alkaline pH on the Sebia Hydrasys 2, and capillary electrophoresis on the Sebia Capillarys 2. Sequencing of the beta globin gene was also conducted. Results: HbA1c analysis by immunoassay gave significantly higher results, ranging from 5.2–5.5%. Hemoglobin fractionation by HPLC showed an abnormal peak comprising approximately 43% of total hemoglobin, suggesting the presence of a beta chain hemoglobin variant. Gel electrophoresis at alkaline pH revealed a very unusual pattern, with 3 abnormal bands migrating with Hb F, between Hb F and Hb S, and slightly cathodal to Hb S. A single band in the Hb A position was seen on gel electrophoresis at acid pH. Capillary electrophoresis revealed two abnormal peaks, comprising 42% and 5% of total hemoglobin. Sequencing of the beta globin gene showed heterozygosity for Hb Hirose (beta 37(C3) Trp N Ser), an extremely rare variant with a substitution at the α1β2 interface. Conclusions: We describe the chromatographic and electrophoretic properties of Hb Hirose, and demonstrate that this rare variant causes falsely low HbA1c results on the BioRad variant II Turbo 2.0. Recognition of this interference is crucial in order to prevent reporting erroneous results. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
1. Introduction Hemoglobin A1c (HbA1c) is a chemically modified form of hemoglobin in which a glucose molecule is covalently bound to the N-terminus of the β-globin chain. As such, HbA1c concentration in whole blood reflects glycemic control over the lifespan of the red blood cell (RBC). Traditionally, HbA1c measurements were used to monitor long-term glycemic control in patients with diabetes mellitus. However, current clinical practice guidelines also endorse the use of HbA1c as a diagnostic marker, with concentrations ≥6.5% being consistent with type 2 diabetes mellitus (T2DM) [1]. HbA1c has several advantages over fasting glucose, including superior analyte stability and no requirement for patient fasting [2]. However, HbA1c measurement is not free of limitations. Disorders of altered RBC turnover may change the relationship between blood glucose and HbA1c concentrations [2,3]. Consequently, patients with ⁎ Corresponding author at: DynaLIFEDx, #200, 10150-102 Street, Edmonton, Alberta T5J 5E2, Canada. E-mail address: [email protected] (M.P. Estey).
such disorders may have spurious HbA1c results independent of the analytical method used. The presence of hemoglobin variants may also interfere with HbA1c analysis, with the degree and direction of interference depending on both the analytical method and the identity of the particular variant [2–6]. In this study, we investigate the cause of low HbA1c results in three siblings being screened for T2DM. There was no evidence of a disorder altering red blood cell lifespan. However, hemoglobinopathy investigation revealed heterozygosity for hemoglobin Hirose, an extremely rare β-globin variant with a substitution at the α1β2 interface. We describe the HPLC, gel electrophoresis, and capillary electrophoresis patterns of Hb Hirose and demonstrate that this rare variant negatively interferes with HbA1c measurement on the BioRad Variant II Turbo 2.0.
2. Materials and methods All analyses were performed on EDTA anti-coagulated whole blood according to the manufacturer's instructions. This study complied with institutional ethics requirements.
http://dx.doi.org/10.1016/j.clinbiochem.2015.11.016 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: M.P. Estey, et al., Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.11.016
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M.P. Estey et al. / Clinical Biochemistry xxx (2015) xxx–xxx
2.1. HbA1c analysis HbA1c was measured by ion exchange chromatography on the BioRad Variant II Turbo 2.0 (analysis time 1.5 min) and by immunoassay using the Siemens DCA 2000+. 2.2. Chromatographic separation of human hemoglobin Hemoglobin fractionation was performed by HPLC using the β-thalassemia Short Program on the BioRad Variant II, as described previously [7]. The difference in resolution between the β-thalassemia method and the HbA1c method is primarily due to differences in buffer composition, gradient profiles, and separation time. Each method has been optimized to achieve different analytical goals (HbA1c quantitation versus Hb A2/Hb F quantitation and qualitative Hb variant information). 2.3. Electrophoresis Agarose gel electrophoresis was performed at acid (6.5) and alkaline (8.6) pH using the Sebia Hydrasys 2 system, as previously described [8]. Capillary electrophoresis was conducted on the Sebia Capillarys 2. 2.4. DNA sequencing Sequencing of the beta globin gene was conducted at the Molecular Genetics Laboratory, Laboratory Reference Centre, Hamilton Regional Laboratory Medicine Program (LRC-HRLMP), McMaster University, Hamilton, Canada.
abnormal peak quantitating at 5% was also noted (identified by the instrument as ‘degraded hemoglobin’). This peak could correspond to one or both of the faint bands seen on gel electrophoresis at alkaline pH. Review of hemoglobin variant databases ([9,10] and the BioRad library of variants) in addition to a thorough literature search did not uncover any hemoglobin variant with chromatographic and electrophoretic properties matching those described above. As a result, sequencing of the beta globin gene was performed, which indicated heterozygosity for an extremely rare variant called Hb Hirose (beta 37(C3) Trp N Ser). 4. Discussion Hb Hirose was first identified in a family from Japan in 1971 [11], and was subsequently found in two unrelated Japanese patients in 1983 [12]. To our knowledge, these are the only reports of patients having this hemoglobin variant. As such, data on the chromatographic and electrophoretic properties of Hb Hirose using modern techniques is lacking. Intriguingly, the siblings described in this report are from Trinidad and Tobago, with no known Japanese ancestry. This raises the possibility that the Hb Hirose mutation may have arisen on more than one occasion. The tryptophan residue at position 37 of the β-globin chain is located at the α1β2 interface of the hemoglobin tetramer, where it plays an important role in holding the two αβ dimers together [13]. The tryptophan to serine mutation in Hb Hirose is thought to destabilize this interface. As a result, Hb Hirose has a tendency to dissociate into subunits, which likely accounts for the multiple bands observed on electrophoresis at alkaline pH (Fig. 2A). Despite this property, patients with Hb Hirose trait, including those described here, do not exhibit any clinically significant symptoms [11,12].
3. Results 4.1. Falsely low HbA1c results in the presence of Hb variants 3.1. HbA1c analysis HbA1c was measured for routine screening purposes in three siblings with a family history of T2DM. HPLC analysis using the BioRad Variant II gave low results for all three patients, ranging from 3.3– 3.5%. They had normal fasting glucose (ranging from 4.6–5.2 mmol/L), unremarkable complete blood counts, and no evidence of a disorder causing increased RBC turnover. Inspection of the HPLC chromatograms showed an abnormal peak with the same retention time as HbA1c (representative example shown in Fig. 1A). We therefore hypothesized that a hemoglobin variant could be interfering with the HbA1c analysis. Consistent with this possibility, HbA1c measurement using an alternate method based on a different analytical principle (the Siemens DCA 2000+ immunoassay) gave significantly higher results, ranging from 5.2–5.5%.
One of the major caveats of HbA1c measurement is the potential for interference from hemoglobin variants [2,3,5,6]. In chromatographic methods, variants that co-elute with the HbA1c fraction cause falsely elevated HbA1c. For example, we recently described a patient with Hb Wayne trait who was misdiagnosed with type 2 diabetes based on falsely elevated HbA1c results [14,15]. It is important to note that hemoglobin variants can also cause falsely decreased HbA1c results, as described here. The BioRad Variant II Turbo 2.0 HbA1c chromatogram (Fig. 1A) showed 87% Hb Ao and no large peak corresponding to a Hb variant, suggesting that Hb Ao and Hb Hirose co-elute using this method. However, the chromatogram did show an abnormal peak directly behind the HbA1c peak, which likely corresponds to glycated Hb Hirose. Chromatographic methods calculate the % HbA1c as follows [5]: %HbA1c ¼ 100 HbA1c=ðHbA1c þ Hb AoÞ:
3.2. Hemoglobinopathy investigation To further investigate the presence of a hemoglobin variant, hemoglobin fractionation was performed by HPLC and electrophoresis. The HPLC chromatogram showed an abnormal peak comprising 43% of total hemoglobin at a retention time of 2.30 min., consistent with the presence of a beta chain hemoglobin variant (Fig. 1B). Gel electrophoresis at alkaline pH revealed a very unusual pattern, with three abnormal bands noted (Fig. 2A, lanes 1–3): a large band migrating slightly cathodal to Hb S, a faint band migrating in the Hb F position, and another faint band migrating between Hb F and Hb S. A single band in the Hb A position was seen on gel electrophoresis at acid pH (Fig. 2B, lanes 1–3). Consistent with the HPLC results, capillary electrophoresis showed an abnormal peak comprising 42% of total hemoglobin (Fig. 2C). A second
While the abnormal peak and the HbA1c peak have the same retention time, the software appears to have excluded the abnormal peak area from the quantitation of the HbA1c peak (Fig. 1A). Thus, glycated Hb Hirose is likely not taken into account in the numerator of the above equation. Since unglycated Hb Hirose co-elutes with Hb Ao, the denominator is overestimated, yielding falsely low HbA1c results. A similar phenomenon has been described for other hemoglobin variants, with the degree of interference being method-dependent [4,5]. The National Academy of Clinical Biochemistry (NACB) recommends that all HbA1c results below the lower limit of the reference interval should be verified by repeat analysis [3]. Since the effect of a particular hemoglobin variant on HbA1c results is often method dependent, repeat analysis should ideally be performed using a method based on
Fig. 1. HPLC analysis reveals low HbA1c and the presence of a beta chain hemoglobin variant. Hb fractionation was performed on the BioRad Variant II using the HbA1c program (A) and the β-thalassemia short program (B). The arrow in panel (A) points to the abnormal Hb peak.
Please cite this article as: M.P. Estey, et al., Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.11.016
M.P. Estey et al. / Clinical Biochemistry xxx (2015) xxx–xxx
Please cite this article as: M.P. Estey, et al., Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.11.016
3
4
M.P. Estey et al. / Clinical Biochemistry xxx (2015) xxx–xxx
Fig. 2. Electrophoretic examination of the Hb variant. Gel electrophoresis was performed at alkaline (A) and acid (B) pH using the Sebia Hydrasys 2 system. Specimens from the three siblings were run in lanes 1–3, whereas lane 4 shows a patient with Hb S trait. (C) Capillary electrophoresis was performed on the Sebia Capillarys 2. The Hb fractions quantitated as follows: Degraded Hb 4.9%, Hb A 50.1%, Hb X 42.3%, and Hb A2 2.7%.
a different analytical principle than the initial assay. Discordant results between methods point towards the presence of a hemoglobin variant, as illustrated here. However, it must be stressed that a HbA1c result within the reference interval does not rule out negative interference from a hemoglobin variant. As such, inspection of the HPLC chromatogram for abnormal peaks is also crucial to identify erroneous HbA1c results due to the presence of hemoglobin variants. 5. Conclusions We describe the chromatographic and electrophoretic properties of Hb Hirose using modern methodologies, which will facilitate the identification of this rare hemoglobin variant by diagnostic laboratories. In addition, we demonstrate that Hb Hirose causes falsely low HbA1c results by HPLC. Recognition of this interference is critical, as reporting falsely low HbA1c results could potentially lead to missed diabetes diagnoses and under treatment of known diabetics. Acknowledgments The authors thank DynaLIFEDx medical technologists for all their help and technical support, and Sylvain Lampron for facilitating the capillary electrophoresis analysis. References [1] J.M. Ekoe, Z. Punthakee, T. Ransom, A.P. Prebtani, R. Goldenberg, Screening for type 1 and type 2 diabetes, Can. J. Diabetes 37 (Suppl. 1) (Apr 2013) S12-5. [2] T. Higgins, HbA1c for screening and diagnosis of diabetes mellitus, Endocrine 43 (2) (Apr 2013) 266–273.
[3] D.B. Sacks, M. Arnold, G.L. Bakris, D.E. Bruns, A.R. Horvath, M.S. Kirkman, et al., Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus, Diabetes Care 34 (6) (Jun 2011) e61–e99. [4] A.C. Bergman, S. Beshara, I. Byman, R. Karim, B. Landin, A new beta-chain variant: Hb stockholm [beta 7(A4)GluAsp] causes falsely low Hb A(1c), Hemoglobin 33 (2) (2009) 137–142. [5] L. Bry, P.C. Chen, D.B. Sacks, Effects of hemoglobin variants and chemically modified derivatives on assays for glycohemoglobin, Clin. Chem. 47 (2) (Feb 2001) 153–163. [6] R.R. Little, S.L. La'ulu, S.E. Hanson, C.L. Rohlfing, R.L. Schmidt, Effects of 49 different rare Hb variants on HbA1c measurement in eight methods, J. Diabetes Sci. Technol. 9 (4) (Jul 2015) 849–856. [7] M.P. Estey, M. Belletrutti, K. Rodriguez-Capote, T. Higgins, Hemoglobin constant spring exhibits prolonged ex vivo stability when assessed by HPLC, Clin. Biochem. 48 (10–11) (Jul 2015) 709–712. [8] K. Rodriguez-Capote, M.P. Estey, V. Barakauskas, P. Bordeleau, C.L. Christensen, P. Zuberbuhler, et al., A novel double heterozygous Hb Fontainebleau/HbD Punjab hemoglobinopathy, Clin. Biochem. 48 (13–14) (Sept 2015) 904–907. [9] B.J. Bain, B.J. Wild, A.D. Stephens, L. Phelan, Variant Haemoglobins; a Guide to Identification, Wiley-Blackwell, Oxford, 2010. [10] J. Szuberski, J.L. Oliveira, J.D. Hoyer, A comprehensive analysis of hemoglobin variants by high-performance liquid chromatography (HPLC), Int. J. Lab. Hematol. 20 (Jun 2012). [11] K. Yamaoka, Hemoglobin Hirose: α2β2 37(C3) tryptophan yielding serine, Blood 38 (6) (Dec 1971) 730–738. [12] Y. Ohba, Y. Hattori, K. Fuyuno, I. Takeda, M. Matsuoka, H. Yoshinaka, et al., Two further examples of Hb Hirose, beta 37 (C3) Trp—-Ser, Hemoglobin 7 (2) (1983) 191–193. [13] J. Sasaki, T. Imamura, T. Yanase, Hemoglobin Hirose, a human hemoglobin variant with a substitution at the alpha1beta2 interface. Subunit dissociation and the equilibria and kinetics of ligand binding, J. Biol. Chem. 253 (1) (Jan 10 1978) 87–94. [14] K. Rodriguez-Capote, M.P. Estey, V.E. Barakauskas, T. Burton, D. Holmes, R. Krause, et al., Identification of Hb Wayne and its effects on HbA1c measurement by 5 methods, Clin. Biochem. 48 (16–17) (Nov 2015) 1144–1150. [15] E. Turley, K. Rodriguez-Capote, M.P. Estey, V.E. Barakauskas, G.R. Bailey, L.M. McKenzie, et al., Erroneous diabetes diagnosis: a case of HbA1c interference, Diabetes Care 20 (Aug 2015).
Please cite this article as: M.P. Estey, et al., Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.11.016
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Short Communication
Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC Mathew P. Estey a,b,⁎, Karina Rodriguez-Capote a,b, Tolulope Adelowokan c, Trefor Higgins a a b c
DynaLIFEDx, Edmonton, Canada Department of Laboratory Medicine and Pathology, University of Alberta, Canada Synergy Medical Clinic, Sherwood Park, Canada
a r t i c l e
i n f o
Article history: Received 22 September 2015 Received in revised form 19 November 2015 Accepted 20 November 2015 Available online xxxx Keywords: Hemoglobin Hirose HbA1c High performance liquid chromatography Electrophoresis Hemoglobinopathy
a b s t r a c t Objectives: To investigate the underlying cause of unexpectedly low HbA1c results (3.3–3.5%) obtained by HPLC in three siblings undergoing routine screening for type 2 diabetes mellitus. Design and methods: HbA1c was measured using an alternate method based on a different analytical principle (the Siemens DCA 2000+ immunoassay). Hemoglobin fractionation was performed by HPLC on the BioRad Variant II, gel electrophoresis at acid and alkaline pH on the Sebia Hydrasys 2, and capillary electrophoresis on the Sebia Capillarys 2. Sequencing of the beta globin gene was also conducted. Results: HbA1c analysis by immunoassay gave significantly higher results, ranging from 5.2–5.5%. Hemoglobin fractionation by HPLC showed an abnormal peak comprising approximately 43% of total hemoglobin, suggesting the presence of a beta chain hemoglobin variant. Gel electrophoresis at alkaline pH revealed a very unusual pattern, with 3 abnormal bands migrating with Hb F, between Hb F and Hb S, and slightly cathodal to Hb S. A single band in the Hb A position was seen on gel electrophoresis at acid pH. Capillary electrophoresis revealed two abnormal peaks, comprising 42% and 5% of total hemoglobin. Sequencing of the beta globin gene showed heterozygosity for Hb Hirose (beta 37(C3) Trp N Ser), an extremely rare variant with a substitution at the α1β2 interface. Conclusions: We describe the chromatographic and electrophoretic properties of Hb Hirose, and demonstrate that this rare variant causes falsely low HbA1c results on the BioRad variant II Turbo 2.0. Recognition of this interference is crucial in order to prevent reporting erroneous results. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
1. Introduction Hemoglobin A1c (HbA1c) is a chemically modified form of hemoglobin in which a glucose molecule is covalently bound to the N-terminus of the β-globin chain. As such, HbA1c concentration in whole blood reflects glycemic control over the lifespan of the red blood cell (RBC). Traditionally, HbA1c measurements were used to monitor long-term glycemic control in patients with diabetes mellitus. However, current clinical practice guidelines also endorse the use of HbA1c as a diagnostic marker, with concentrations ≥6.5% being consistent with type 2 diabetes mellitus (T2DM) [1]. HbA1c has several advantages over fasting glucose, including superior analyte stability and no requirement for patient fasting [2]. However, HbA1c measurement is not free of limitations. Disorders of altered RBC turnover may change the relationship between blood glucose and HbA1c concentrations [2,3]. Consequently, patients with ⁎ Corresponding author at: DynaLIFEDx, #200, 10150-102 Street, Edmonton, Alberta T5J 5E2, Canada. E-mail address: [email protected] (M.P. Estey).
such disorders may have spurious HbA1c results independent of the analytical method used. The presence of hemoglobin variants may also interfere with HbA1c analysis, with the degree and direction of interference depending on both the analytical method and the identity of the particular variant [2–6]. In this study, we investigate the cause of low HbA1c results in three siblings being screened for T2DM. There was no evidence of a disorder altering red blood cell lifespan. However, hemoglobinopathy investigation revealed heterozygosity for hemoglobin Hirose, an extremely rare β-globin variant with a substitution at the α1β2 interface. We describe the HPLC, gel electrophoresis, and capillary electrophoresis patterns of Hb Hirose and demonstrate that this rare variant negatively interferes with HbA1c measurement on the BioRad Variant II Turbo 2.0.
2. Materials and methods All analyses were performed on EDTA anti-coagulated whole blood according to the manufacturer's instructions. This study complied with institutional ethics requirements.
http://dx.doi.org/10.1016/j.clinbiochem.2015.11.016 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: M.P. Estey, et al., Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.11.016
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M.P. Estey et al. / Clinical Biochemistry xxx (2015) xxx–xxx
2.1. HbA1c analysis HbA1c was measured by ion exchange chromatography on the BioRad Variant II Turbo 2.0 (analysis time 1.5 min) and by immunoassay using the Siemens DCA 2000+. 2.2. Chromatographic separation of human hemoglobin Hemoglobin fractionation was performed by HPLC using the β-thalassemia Short Program on the BioRad Variant II, as described previously [7]. The difference in resolution between the β-thalassemia method and the HbA1c method is primarily due to differences in buffer composition, gradient profiles, and separation time. Each method has been optimized to achieve different analytical goals (HbA1c quantitation versus Hb A2/Hb F quantitation and qualitative Hb variant information). 2.3. Electrophoresis Agarose gel electrophoresis was performed at acid (6.5) and alkaline (8.6) pH using the Sebia Hydrasys 2 system, as previously described [8]. Capillary electrophoresis was conducted on the Sebia Capillarys 2. 2.4. DNA sequencing Sequencing of the beta globin gene was conducted at the Molecular Genetics Laboratory, Laboratory Reference Centre, Hamilton Regional Laboratory Medicine Program (LRC-HRLMP), McMaster University, Hamilton, Canada.
abnormal peak quantitating at 5% was also noted (identified by the instrument as ‘degraded hemoglobin’). This peak could correspond to one or both of the faint bands seen on gel electrophoresis at alkaline pH. Review of hemoglobin variant databases ([9,10] and the BioRad library of variants) in addition to a thorough literature search did not uncover any hemoglobin variant with chromatographic and electrophoretic properties matching those described above. As a result, sequencing of the beta globin gene was performed, which indicated heterozygosity for an extremely rare variant called Hb Hirose (beta 37(C3) Trp N Ser). 4. Discussion Hb Hirose was first identified in a family from Japan in 1971 [11], and was subsequently found in two unrelated Japanese patients in 1983 [12]. To our knowledge, these are the only reports of patients having this hemoglobin variant. As such, data on the chromatographic and electrophoretic properties of Hb Hirose using modern techniques is lacking. Intriguingly, the siblings described in this report are from Trinidad and Tobago, with no known Japanese ancestry. This raises the possibility that the Hb Hirose mutation may have arisen on more than one occasion. The tryptophan residue at position 37 of the β-globin chain is located at the α1β2 interface of the hemoglobin tetramer, where it plays an important role in holding the two αβ dimers together [13]. The tryptophan to serine mutation in Hb Hirose is thought to destabilize this interface. As a result, Hb Hirose has a tendency to dissociate into subunits, which likely accounts for the multiple bands observed on electrophoresis at alkaline pH (Fig. 2A). Despite this property, patients with Hb Hirose trait, including those described here, do not exhibit any clinically significant symptoms [11,12].
3. Results 4.1. Falsely low HbA1c results in the presence of Hb variants 3.1. HbA1c analysis HbA1c was measured for routine screening purposes in three siblings with a family history of T2DM. HPLC analysis using the BioRad Variant II gave low results for all three patients, ranging from 3.3– 3.5%. They had normal fasting glucose (ranging from 4.6–5.2 mmol/L), unremarkable complete blood counts, and no evidence of a disorder causing increased RBC turnover. Inspection of the HPLC chromatograms showed an abnormal peak with the same retention time as HbA1c (representative example shown in Fig. 1A). We therefore hypothesized that a hemoglobin variant could be interfering with the HbA1c analysis. Consistent with this possibility, HbA1c measurement using an alternate method based on a different analytical principle (the Siemens DCA 2000+ immunoassay) gave significantly higher results, ranging from 5.2–5.5%.
One of the major caveats of HbA1c measurement is the potential for interference from hemoglobin variants [2,3,5,6]. In chromatographic methods, variants that co-elute with the HbA1c fraction cause falsely elevated HbA1c. For example, we recently described a patient with Hb Wayne trait who was misdiagnosed with type 2 diabetes based on falsely elevated HbA1c results [14,15]. It is important to note that hemoglobin variants can also cause falsely decreased HbA1c results, as described here. The BioRad Variant II Turbo 2.0 HbA1c chromatogram (Fig. 1A) showed 87% Hb Ao and no large peak corresponding to a Hb variant, suggesting that Hb Ao and Hb Hirose co-elute using this method. However, the chromatogram did show an abnormal peak directly behind the HbA1c peak, which likely corresponds to glycated Hb Hirose. Chromatographic methods calculate the % HbA1c as follows [5]: %HbA1c ¼ 100 HbA1c=ðHbA1c þ Hb AoÞ:
3.2. Hemoglobinopathy investigation To further investigate the presence of a hemoglobin variant, hemoglobin fractionation was performed by HPLC and electrophoresis. The HPLC chromatogram showed an abnormal peak comprising 43% of total hemoglobin at a retention time of 2.30 min., consistent with the presence of a beta chain hemoglobin variant (Fig. 1B). Gel electrophoresis at alkaline pH revealed a very unusual pattern, with three abnormal bands noted (Fig. 2A, lanes 1–3): a large band migrating slightly cathodal to Hb S, a faint band migrating in the Hb F position, and another faint band migrating between Hb F and Hb S. A single band in the Hb A position was seen on gel electrophoresis at acid pH (Fig. 2B, lanes 1–3). Consistent with the HPLC results, capillary electrophoresis showed an abnormal peak comprising 42% of total hemoglobin (Fig. 2C). A second
While the abnormal peak and the HbA1c peak have the same retention time, the software appears to have excluded the abnormal peak area from the quantitation of the HbA1c peak (Fig. 1A). Thus, glycated Hb Hirose is likely not taken into account in the numerator of the above equation. Since unglycated Hb Hirose co-elutes with Hb Ao, the denominator is overestimated, yielding falsely low HbA1c results. A similar phenomenon has been described for other hemoglobin variants, with the degree of interference being method-dependent [4,5]. The National Academy of Clinical Biochemistry (NACB) recommends that all HbA1c results below the lower limit of the reference interval should be verified by repeat analysis [3]. Since the effect of a particular hemoglobin variant on HbA1c results is often method dependent, repeat analysis should ideally be performed using a method based on
Fig. 1. HPLC analysis reveals low HbA1c and the presence of a beta chain hemoglobin variant. Hb fractionation was performed on the BioRad Variant II using the HbA1c program (A) and the β-thalassemia short program (B). The arrow in panel (A) points to the abnormal Hb peak.
Please cite this article as: M.P. Estey, et al., Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.11.016
M.P. Estey et al. / Clinical Biochemistry xxx (2015) xxx–xxx
Please cite this article as: M.P. Estey, et al., Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.11.016
3
4
M.P. Estey et al. / Clinical Biochemistry xxx (2015) xxx–xxx
Fig. 2. Electrophoretic examination of the Hb variant. Gel electrophoresis was performed at alkaline (A) and acid (B) pH using the Sebia Hydrasys 2 system. Specimens from the three siblings were run in lanes 1–3, whereas lane 4 shows a patient with Hb S trait. (C) Capillary electrophoresis was performed on the Sebia Capillarys 2. The Hb fractions quantitated as follows: Degraded Hb 4.9%, Hb A 50.1%, Hb X 42.3%, and Hb A2 2.7%.
a different analytical principle than the initial assay. Discordant results between methods point towards the presence of a hemoglobin variant, as illustrated here. However, it must be stressed that a HbA1c result within the reference interval does not rule out negative interference from a hemoglobin variant. As such, inspection of the HPLC chromatogram for abnormal peaks is also crucial to identify erroneous HbA1c results due to the presence of hemoglobin variants. 5. Conclusions We describe the chromatographic and electrophoretic properties of Hb Hirose using modern methodologies, which will facilitate the identification of this rare hemoglobin variant by diagnostic laboratories. In addition, we demonstrate that Hb Hirose causes falsely low HbA1c results by HPLC. Recognition of this interference is critical, as reporting falsely low HbA1c results could potentially lead to missed diabetes diagnoses and under treatment of known diabetics. Acknowledgments The authors thank DynaLIFEDx medical technologists for all their help and technical support, and Sylvain Lampron for facilitating the capillary electrophoresis analysis. References [1] J.M. Ekoe, Z. Punthakee, T. Ransom, A.P. Prebtani, R. Goldenberg, Screening for type 1 and type 2 diabetes, Can. J. Diabetes 37 (Suppl. 1) (Apr 2013) S12-5. [2] T. Higgins, HbA1c for screening and diagnosis of diabetes mellitus, Endocrine 43 (2) (Apr 2013) 266–273.
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Please cite this article as: M.P. Estey, et al., Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.11.016