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| Year : 2015 | Volume
: 58
| Issue : 4 | Page : 572-574 |
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| Interference of hemoglobin D Punjab on measurements of glycated hemoglobin |
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Anju Shukla1, Sunil Dabadghao2, Surbhi Gupta1, Sunil Verma3
1 Department of Laboratory Medicine, Sahara Hospital, Lucknow, Uttar Pradesh, India
2 Department of Haematology, Sahara Hospital, Lucknow, Uttar Pradesh, India
3 Department of Medicine, Sahara Hospital, Lucknow, Uttar Pradesh, India
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| Date of Web Publication | 4-Nov-2015 |
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How to cite this article:
Shukla A, Dabadghao S, Gupta S, Verma S. Interference of hemoglobin D Punjab on measurements of glycated hemoglobin. Indian J Pathol Microbiol 2015;58:572-4 |
How to cite this URL:
Shukla A, Dabadghao S, Gupta S, Verma S. Interference of hemoglobin D Punjab on measurements of glycated hemoglobin. Indian J Pathol Microbiol [serial online] 2015 [cited 2023 Sep 29];58:572-4. Available from: https://www.ijpmonline.org/text.asp?2015/58/4/572/168874 |
A 55-year-old Punjabi man had come for routine advanced health check-up in our hospital. On examination, his liver and kidney function tests were within normal limit, lipid profile revealed increased serum cholesterol: 237 mg/dl increased serum triglyceride: 263 mg/dl, and low high-density lipoprotein cholesterol: 35 mg/dl. His uric acid was raised: 8.10 mg/dl. Fasting glucose was 109 mg/dl, while glycated hemoglobin (HbA1c) was 9.8%. Complete blood count showed Hb: 15.2 g/dl, total leucocyte count: 5000 cells/cumm, differential leukocytic count: P55%L40%E04%M01%, platelet count: 260,000/cumm. Urine routine and microscopic examination and serum prostate-specific antigen were within normal limit. On interpretation of results, we found that there is a discrepancy between normal fasting glucose level and increased HbA1c percentage. HbA1C was performed by high-performance liquid chromatography (HPLC) on Bio-Rad D-10 instrument using short program, while plasma glucose was estimated by enzymatic reference method with hexokinase on Cobas Integra 400 plus testing system. Bio-Rad D-10 Hb testing system has two modes; one short program mode for percent determination of HbA1C, and extended program mode for percent determination of HbA1C, HbF, and HbA2. On careful examination of chromatogram, it was found that there is a large variant window at the retention time of 1.63 min covering 91.8% area [Figure 1]. On correlation of peak with their retention time on short program, it is seen that S window generally elute at retention time of 1.67 min, while unknown peak in our case was present at retention time of 1.63 min. | Figure 1: Chromatograph of glycated hemoglobin on high--performance liquid chromatography system (short program) showing variant window at retention time of 1.63 minHbA2.
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Sample was again analyzed on Bio-Rad D-10 system, this time utilizing extended program mode and the chromatogram revealed an unknown window at the retention time of 3.92 min [Figure 2]. On correlation of peak with their retention time on extended program, it is seen that S window generally elute at retention time of 4.20 min, while unknown peak in our case was at retention time of 3.92 min. On review of literature, it was found that HbD Punjab elutes in an unknown window at a retention time of approximately 3.8 min clearly demarcated from HbS peak.[1] | Figure 2: Chromatograph on glycated hemoglobin (extended program) showing variant window at retention time of 3.92 min
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Sample was further analyzed for Hb electrophoresis cellulose acetate at pH 8.6, and a distinct band was obtained at S/D region, which migrate with adult Hb on citrate agar electrophoresis at pH 6.0.
Complete blood count with reticulocyte count was repeated and was found normal. Sickling test was negative. On examination, there was no organomegaly or lymphadenopathy, and there was no sign of hemolysis.
Therefore with distinct ethnicity, normal blood count, no sign of hemolysis, distinct band at S/D/G area on alkaline electrophoresis, which moved with adult Hb on acid electrophoresis and a separate window on HPLC, clearly demarcated from the HbS peak, a probable diagnosis of HbD Punjab was made and sample was sent to referral laboratory for molecular diagnosis and confirmation.
Meanwhile, HbA1C was estimated by immunoassay method using Tina-quant HbA1C Gen. 2 reagent on Cobas Integra 400 plus instrument (Roche Diagnostics) and the result obtained was 5.4%.
Molecular diagnosis with gene sequencing studies confirms the diagnosis of HbD Punjab.
Therefore, it was concluded that Hb variant (HbD) cannot be separated from HbA1C on HPLC and co-elution of Hb variant with HbA1C causes overestimation of HbA1C.
 Discussion | |  |
The most common Hb variants in decreasing order of frequency worldwide are HbS, HbE, HbC, and HbD. All of these Hbs have single amino acid substitution in the β chain.
Hemoglobin S homozygous cause moderate to severe anemia, irreversible sickle cells, and characteristic HPLC pattern; large S window with increased HbF. HbS heterozygous have normal Hb and on HPLC, HbS ranges between 30% and 40% in S window at retention time of approximately 4.20 s.
Hemoglobin E (both homozygous and heterozygous) elutes in HbA2 window and may split to give an additional peak just before the HbA2 peak.
Hemoglobin C trait is clinically silent and shows HbC ranging between 30% and 40% in C window.
Clinically silent Hb variants are more challenging during interpretation of HbA1C chromatographs. The presence of HbS or the HbC trait has been shown to affect the accuracy of some HbA1C assay by previous reports [2] however effect of HbE on HbA1C measurement has not been well-documented.
Hemoglobin D, which is observed in our case, is the fourth most common Hb variant worldwide and caused by β chain mutation at position 121, where glutamine replaces glutamic acid. Four forms of HbD exist (A-D):
- Heterozygous HbD trait, where HbD is about 30–40% and the condition is clinically silent. In the Punjab region, this heterozygosity reaches about 3%
- Homozygous HbD disease, where HbD is about 80–90%, but 95% of homozygous HbD have normal red cell indices, no evidence of hemolysis and have normal HbF and HbA2 levels
- HbD-Thalassemia causes mild hemolytic anemia, thalassemic red cell indices, and increased HbF and HbA2 levels
- Hemoglobin S-D disease causes moderate to severe sickling disorder.
It is common for both homozygous and heterozygous HbD patients to have normal blood counts but there HbA1C can be wrongly interpreted at times and does not give the correct picture in case of diagnosis and monitoring of diabetes. In our case due to interference of HbD, results obtained for HbA1C are falsely elevated, which is contrary to previous reports where HbA1C value was artificially low.[3],[4]
| Conclusion | | |
Knowledge and awareness of Hb variants affecting HbA1c measurements are essential, especially in areas with a high prevalence of hemoglobinopathy, in order to avoid mismanagement of diabetic patients. To avoid reporting of inaccurate results, it is mandatory that all ion-exchange chromatograms must be carefully examined to identify possible interference from Hb variants and any sample with a combined area of more than 60% in any window HbA1C results should not be reported.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
| References | | |
| 1. | Zeng YT, Huang SZ, Zhou LD, Huang HJ, Jiao CT, Tang ZG, et al. Identification of hemoglobin D Punjab by gene mapping. Hemoglobin 1986;10:87-90.  [ PUBMED] |
| 2. | Mongia SK, Little RR, Rohlfing CL, Hanson S, Roberts RF, Owen WE, et al. Effects of hemoglobin C and S traits on the results of 14 commercial glycated hemoglobin assays. Am J Clin Pathol 2008;130:136-40. |
| 3. | Little RR, Rohlfing CL, Hanson S, Connolly S, Higgins T, Weykamp CW, et al. Effects of hemoglobin (Hb) E and HbD traits on measurements of glycated Hb (HbA1c) by 23 methods. Clin Chem 2008;54:1277-82. |
| 4. | Schnedl WJ, Lahousen T, Wallner SJ, Krause R, Lipp RW. Silent hemoglobin variants and determination of HbA (1c) with the high-resolution program of the HPLC HA-8160 hemoglobin analyzer. Clin Biochem 2005;38:88-91. |
Correspondence Address:
Dr. Anju Shukla
Department of Laboratory Medicine, Sahara Hospital, Viraj Khand, Gomtinagar, Lucknow, Uttar Pradesh
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0377-4929.168874
[Figure 1], [Figure 2] |
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