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  Table of Contents    
Year : 2013  |  Volume : 56  |  Issue : 4  |  Page : 483-484
Limitation of HPLC methodology for HBA1c estimation

1 Hematology Department, Super Religare Laboratories (Formerly SRL Ranbaxy Pvt Ltd), Clinical Reference Lab, Gurgaon, Haryana, India
2 Biochemistry Department, Super Religare Laboratories (Formerly SRL Ranbaxy Pvt Ltd), Clinical Reference Lab, Gurgaon, Haryana, India

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Date of Web Publication18-Jan-2014

How to cite this article:
Dam AR, Ahuja AJ, Singh M, Singh R. Limitation of HPLC methodology for HBA1c estimation. Indian J Pathol Microbiol 2013;56:483-4

How to cite this URL:
Dam AR, Ahuja AJ, Singh M, Singh R. Limitation of HPLC methodology for HBA1c estimation. Indian J Pathol Microbiol [serial online] 2013 [cited 2021 Jun 17];56:483-4. Available from: https://www.ijpmonline.org/text.asp?2013/56/4/483/125413


The American Diabetes Association (ADA) recommends HBA1c as the standard laboratory assessment of glycemic control and efficacy of treatment. Currently, there are five available methods of testing for HbA1c in the laboratory. These methods use separation based on

  • charge differences - ion exchange chromatography, HPLC, electrophoresis, and isoelectric focusing
  • structural differences - affinity chromatography and immunoassay
  • chemical analysis - photometry, spectrophotometry

We report a case of Hb D homozygous patient diagnosed with the help of high pressure liquid chromatography (HPLC) and ancillary inputs with invalid HBA1c value. We received blood sample of a 58-yr-old patient for HBA1c. The HBA1c reported by the D-10 instrument (HPLC short programme) was 3.8% with a variant window of 87.1% at a retention time of 1.63 minutes [Figure 1]. Complete blood counts revealed Hb 10.1 gm/dL, microcytic hypochromic blood picture with anisopoikilocytosis and mild erythrocytosis. The sample was run on Bio-rad variant (HPLC) and revealed a HBD peak of 81.5% at a RT of 4.05 minutes [Figure 2]. A provisional diagnosis of Hb D Punjab homozygous was made and alternative methodology e.g. boronate affinity was recommended for HBA1c estimation in the patient.
Figure 1: HbA1c chromotograph on D10 showing variant window of 87.1% and low HbA1c value of 3.8%

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Figure 2: Hb variant chromotograph on variant II classic showing variant HbD window of 81.5% -HbD homozygous

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HBA1C represents the main fraction of hemoglobin bound to glucose (glycohemoglobin) and is normally present at low levels in red blood cells. In patients with diabetes having normal hemoglobin, HBA1C values strongly correlate with blood glucose level. Because the A1C test is based on normal hemoglobin, hemoglobinopathies can affect the reliability of the test in three ways: (1) altering the normal process of glycation of HbA to A1C, (2) causing an abnormal peak on chromatography, making estimation of A1C unreliable, and (3) making the red blood cell more prone to hemolysis, thereby decreasing the time for glycosylation to occur and producing a falsely low A1C result [1] .

The influence of Hb variants on HBA1c is shown to be method dependent and also greater when HPLC is used. Although D10 belongs to the first generation of dedicated HBA1c systems still the laboratories must be aware of the possibilities of such interference. The chromatographs should be analyzed carefully to detect any variant windows that might be eluting specially in cases of low HBA1c reports. When there is a discrepancy between HBA1c and blood glucose values, conditions that affect the red cell lifespan and possibility of hemoglobinopathy must be investigated [2] . Such samples with clinically silent hemoglobin variants should be analyzed by a second method with a different assay principle preferably boronate affinity or Electrospray Mass Spectrometry for HBA1c. The true HBA1c value in this case was 5.2%.

   References Top

1.Camargo JL, Gross JL. Conditions associated with very low values of glycohaemoglobin measured by an HPLC method. J Clin Pathol 2004;57:346-9.  Back to cited text no. 1
2.Smaldone A. Glycemic control and haemoglobinopathy: When A1c may not be reliable. Diabetes Spectrum 2008;21:46-9.  Back to cited text no. 2

Correspondence Address:
Arpita R Dam
D 216, Sarita Vihar, New Delhi - 110 076
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.125413

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