Hemoglobin variants in samples requested for HbA1c
Sharwari Narawade*, N Mahalle, M Mandolkar, S Naik
Affiliation
Deenanath Mageshkar Hospital and Research Center, Erandwane, Pune 411004
Corresponding Author
Sadanand Naik, Deenanath Mageshkar Hospital and Research Center, Erandwane, Pune 411004, Email: sadu_naik@rediffmail.com
Citation
Narawade, S., et al. Hemoglobin variants in samples requested for HbA1c (2019) Int J Hemat Ther 5(1): 16-19.
Copy rights
© 2019 Narawade, S. This is an Open access article distributed under the terms of Creative Commons Attribution 4.0 International License.
Keywords
Glycated hemoglobin; Hemoglobin variants; Electrophoresis
Abstract
To determine the prevalence of hemoglobin variants in Indian diabetic patients attending to our hospital and to offer explanations and advise over the findings to the patients with hemoglobinopathy which is not practiced at present Methods: HbA1c concentrations in whole blood was measured using Bio Rad D10 HPLC analyser. Blood of the patients with hemoglobin variants were subjected to alkaline agarose gel electrophoresis to confirm the status. Hematological indices of these patients measured on Sysmex 3000 were reviewed. Results: HbA1c was measured on 5100 patients. 95 patients were found to have hemoglobinopathy (54 with HbS and 36 other variants) and 3 were found to be homozygous (two Hb S one HbD Punjab). However, their hematological findings were normal. Agarose gel electrophoresis confirmed their variant status. Conclusion: In current scenario, the patients are not informed about their Hb status. We suggest that we need to provide the information on hemoglobin status along with HbA1c results to primary health care providers who can refer to haematologist for necessary advice.
Introduction
Glycohemoglobin, measured as hemoglobin (Hb) A1C or as total GHb, is a good biomarker for assessing long-term glycaemic control in individuals with diabetes mellitus. Genetic variants and chemically modified derivatives of Hb can profoundly affect the accuracy of these measurements and there is considerable variation among commercially available methods. The rate of prevalence of diabetes mellitus (DM) is increasing rapidly worldwide. This rate has more than doubled, reaching 10 % in recent years[1]. This increasing trend of DM prevalence demonstrates the significance of measuring glycated hemoglobin (HbA1c) in controlling glucose levels and also long-term monitoring. HbA1c has also been shown to be directly related to the risk of developing diabetes complications[2]. HbA1c biomarker was recently recommended by the American Diabetes Association for the diagnosis, treatment and control[2-3] indicating the importance of accurate and precise measurement of HbA1c. Herman et al. and others demonstrated that HbA1c levels are different among racial and ethnic groups, and a particular reference point was not appropriate for all ethnic groups due to the presence of hemoglobin variants[4]. HPLC technique also has limitation in accurate measurement of HbA1c.
The aim of this study was to determine the prevalence of hemoglobin variants in Indian diabetic patients attending to our hospital, confirm hemoglobinopathy using alkaline / acid agarose gel electrophoresis or boronate affinity chromatography and then to offer explanations and advise over the findings to the patients which is not being practiced at present
Materials and Methods
5100 patients, over the span of 3 years (June 2015 to June 2018), were referred to the pathology laboratory for HbA1c measurements. Non fasting blood was collected in EDTA vacutainers.
HbA1c was measured on Bio-Rad D-10 analyzer[10]. D-10 is dedicated equipment for measuring HbA1c. D-10 was calibrated using Bio-rad calibrators. The internal quality control samples, normal and abnormal, were run every day. We did not obtain the consent from the patients with hemoglobinopathy for performing electrophoresis, but consent was obtained from treating clinicians. However, we obtained consent to measure HbA1c using boronate affinity chromatography technique in order to offer accurate result. Blood samples showing other haemoglobin variants were not subjected to Hb variant analysis because of the extra cost involved. However, the samples which showed no HbA0 were subjected to Hb variant analysis. Agarose gel electrophoresis using barbital buffer, pH 8.2, was carried out on the blood samples which showed Hb S variants. The hemogram results of these blood samples which were run on xm-sysmax 3000, a fully automated 5-part cell counter, were obtained and reviewed.
Results
Coefficient of variation for normal (5.2%) and abnormal (9.9%) HbA1 c samples are 2.5 and 3.1 % respectively. Out of 5100 samples, 95 samples were found to have hemoglobinopathy on D10 out of which 54 were heterozygous HbS and 36 were other Hb variants and three samples showed the absence of A0. The three homozygous samples were processed on Bio-Rad Variant machine. Two exhibited Hb S and the other HbD Punjab. The presence of HbS variants were further confirmed on (alkaline-barbital buffer) agarose gel-electrophoresis. Retrospectively reviewed hemogram results of these patients displayed by the cell counter did not reveal any abnormalities in the RBC morphology.
Figure 1a: HbA1c Chromatogram of patient displaying S-Window with 38.7% area
Figure 1b: Corresponding Electrophoresis slide of above patient with an Unknown Hb variant, processed along with a sample of normal adult
Figure 2a: HbA1c chromatogram of patient displaying unknown variant with 42.6% area
Figure 2b: Corresponding Electrophoresis slide of above patient with an Unknown Hb variant, processed along with a sample of normal adult
Discussion
Many clinical laboratories use Bio-Rad HPLC D10 system for Hb A1c measurements. Hemoglobinopathies are associated with decreased erythrocyte survival and decreased HbA1c percentages and are frequent in many parts of the world; more than 900 hemoglobin variants have been described, and in humans, possible single-point mutations have been identified[6]. Many hemoglobin variants have minimal clinical significance[7]. However, their presence may cause an increase or decrease in the HbA1c result, depending on the methodology and the properties of the hemoglobin[8]. Hb variant finding is an intrinsic part in measuring HbA1c by Bio Rad D10 which uses cation exchange principle. Hemoglobin S (HbS) is the most common variant worldwide and is consequently found in a significant proportion of diabetic patients who are tested for HbA1c[9]. Subject with variants need to use the knowledge of their Hb variant carrier status in reproductive decision that can minimize the risk for offspring of sickle cell / thalassemia disease. D10 is not capable of measuring elevated HbA2. Therefore, beta-thalassemia carriers were not observed. The blood samples with hemoglobinopathies affect the reliability of test to monitor diabetes. Knowledge about the presence of Hb variant can provide health benefits to the carriers. Amongst African Americans, HbS trait has been shown to be associated with an increased risk of chronic kidney disease [11]. Usually, the automated cell counter doesn’t subject the normal samples for smear preparation, for staining and then microscopic examination. Only the flagged samples are taken up for slide preparation and staining. Therefore, all samples are not studied with respect to cell morphology and a normal hemogram results are delivered to patients. We determined that intimating the diabetic patients with regard to their carrier status shall be appropriate. We decided to develop a plan for handling the incidental finding of HbAS and HbAC so that the carriers can share the results with his or her primary care provider and if required genetic counsellor’s advice can be obtained if he / she want to have children. Many clinical laboratories in Pune use HPLC system for HbA1c measurements and prefer not to report nor discuss Hb variants observed for two reasons;
• Patient can question as to why Hb variant is reported when HbA1c was requested by the physician
• Patient questions about the authenticity of the result
• Asymptomatic nature of carrier status. Therefore, we derived a plan for appropriate clinical management by providing results and authenticity of the findings to health care providers as developed by the National Institute of Diabetes and Digestive Kidney diseases.
Conclusion
These observations are only after reviewing HbA1c chromatograms. There may be many more who are undiagnosed on hematological reports and the one who got HbA1c tested by non-HPLC methods. Hb variant status observed and confirmed need to be informed to the clinicians and the individuals for further action and precautions.
Our advice for patients with hemoglobinopathy.
• If patient is not married, screen the partner for Hb variants if he/she is a carrier, more careful advice be given to them,
• Restrict frequent blood donation and
• Screen all family members for Hb variant. We argue for (a) Careful examination of PBS (b) Confirm Hb variants obtained on HPLC-D 10 by agarose gel electrophoresis and (c) Genetic counselling needs to be a part of treatment and for clinicians to request for confirmation of D-10 findings by other methods .
Competing Interests
The authors have no competing interests
Acknowledgements
The authors thank all our technicians’ staff Swati, Swapnali, Sandeep, Jayashree, Shubhangi and Sneha for her help in processing the samples. In addition, we gratefully acknowledge the help of hematology technicians for making the hemogram reports of the patients available to us.
Abbreviations: Hb- Hemoglobin; GHb- Glycohemoglobin; HPLC -High performance liquid chromatography
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