Case Study: Artifactually Low Hemoglobin A1c in a Patient with High Hemoglobin F
D.P. is a 39-year-old Vietnamese woman with a 4-year history of diabetes. There is no history of diabetic retinopathy or nephropathy. She began taking 12 U of NPH insulin each evening after medical nutrition therapy alone was insufficient to control her blood glucose control. Over the course of her treatment, her HbA1c measure-ments, as determined by an immuno-assay (Bayer DCA-2000), varied from 5.1 to 5.8%.
In February 2000, the laboratory switched from the immunoassay to a cation exchange high-performance liquid chromatography (HPLC) method (Bio-Rad Variant II) for determining HbA1c. Her HbA1c by HPLC was 7.8%. The HbA1c as determined by immunoassay on the same blood specimen was 6.9%. The HPLC results suggested the presence of increased hemoglobin F (HbF), which was found to comprise 19.1% of the total hemoglobin, as measured by cation exchange HPLC (Bio-Rad Variant "Classic"). Other hematological data included a hematocrit of 39%, a mean corpuscular volume (MCV) of 79 fl, a mean corpuscular hemoglobin of 24.0 pg, and a HbA2 of 2.8%.
Methods used to measure HbA1c include electrophoresis, boronate affinity chromatography, immunoassay, and cation exchange HPLC.1 The various methods differ in cost, complexity, and precision. A given laboratory must choose a method based on these factors and on the laboratory's workload.
In addition, the laboratory must recognize that the various methods can be affected differently by the presence of inherited hemoglobin variants. In the case of HPLC or electrophoretic methods, variant hemoglobins can migrate with HbA or HbA1c, preventing quantification of HbA1c.2 For immunoassays, the antibody used in the assay might not recognize variant hemoglobins. This latter situation would go unnoticed by the technologist performing the test, since there is no way of detecting the variant hemoglobin in an immunoassay.
This case illustrates how a variant hemoglobin can interfere with one method of HbA1c measurement but not another. In this case, a high level of HbF resulted in an artificially low HbA1c value when an immunoassay was used for HbA1c measurement. The Bayer DCA-2000 uses an antibody specific for the glycated amino terminus of -globin. Fetal hemoglobin contains -globin, rather than -globin, chains. The amino terminal structure of -globin is sufficiently different from -globin that it is not recognized by the antibody. Only glycated HbA, and not glycated HbF, is detected by the antibody. Because in the DCA-2000 method, HbA1c is expressed as a percentage of total hemoglobin including HbF, the result is an incorrectly low value of HbA1c.
This is a known limitation of immunoassays for HbA1c.3 The product literature for the Bayer DCA-2000 instrument indicates that the assay is unreliable in patients with >10% HbF. In contrast, the HPLC method determines HbA1c levels by comparing the areas of the HbA1c and HbA peaks in the HPLC chromatogram. Because the glycated and nonglycated forms of HbF migrate differently than HbA1c and HbA, the assay is not affected by the presence of high levels of HbF.3 Thus, HPLC is the most accurate method to measure HbA1c levels in patients with high HbF.
It should be noted that most common hemoglobin variants, including hemoglobins S, C, and E, do not interfere with either immunoassay or HPLC methods for HbA1c quanitification.3 They do not interfere with the immunoassay because the amino termini of the -globin chains of these variants are identical to that of the HbA -globin chain (this despite hemoglobins S and C having mutations at the sixth amino acid). Furthermore, most of the less common hemoglobin variants will not interfere with immunoassays, again because the amino termini of their -globin chains are identical to those of HbA. (Exceptions would include variant hemoglobins with mutations very close to the amino terminus.2)
In contrast, those hemoglobin variants that comigrate with HbA1c on HPLC will interfere with HbA1c determination, requiring the use of an immunoassay or boronate affinity method to measure HbA1c. Finally, the red cells of patients with hemoglobins S and C have shorter-than-normal lifespans, so these patients will, on average, have lower HbA1c levels than expected for their actual level of glucose control.4 This will be true for patients who have short red cell lifespans for other reasons, including hemolytic anemia.
D.P.'s HbF is most likely elevated on a genetic basis, i.e., hereditary persistence of fetal hemoglobin (HPFH). HPFH can be due to either deletion of the and globin genes on the affected chromosome 11 or to point mutations in the promoter of one of the -globin genes.5
When inherited with sickle-cell anemia or -thalassemia, HPFH can result in a milder clinical syndrome. HPFH is common in populations that have high frequencies of other hemoglobinopathies and thalassemias, particularly in patients of Mediterranean, African, and Asian origin.
D.P. had a mild microcytosis. This could indicate the presence of coincident thalassemia trait, although iron deficiency should be ruled out before making this diagnosis. D.P.'s first-degree relatives could be studied to determine whether the elevated HbF and microcytosis were inherited. There are some reports in the literature indicating that patients with diabetes have elevated HbF levels, but the reported elevations are minimal and would not affect HbA1c measurement by any assay.6
Because D.P. is now known to have a high HbF level, in the future, her glycemic control should be assessed using either a boronate affinity or cation exchange HPLC method.
This case illustrates another pointthat one cannot know a priori whether a patient's HbA1c levels are accurate, because the patient could be harboring a hemoglobin variant that interferes with immunologic detection of HbA1c. This situation might be suspected if the level of HbA1c is different than would be expected based on the results of a patient's home blood glucose monitoring. If possible, all patients should have at least one HPLC assay for HbA1c to rule out the presence of interfering hemoglobins. Because the National Glycohemoglobin Standardization Program now standardizes most HbA1c methods in use, the HbA1c levels determined by different methodologies should be comparable.
1Goldstein DR, Little RR, Lorenz RA, Malone JI, Nathan D, Peterson CM: Tests of glycemia in diabetes. Diabetes Care 18:896-909, 1995.
2Roberts WL, Frank EL, Moulton L, Papadea C, Noffsinger JK, Ou C-N: Effects of nine hemoglobin variants on five glycohemoglobin methods. Cell Growth Diff 46:569-72, 2000.
3Chang J, Hoke C, Ettinger B, Penerian G: Evaluation and interference study of hemoglobin A1c measured by turbidimetric inhibition immunoassay. Am J Clin Pathol 109:274-78, 1998.
4Martina WV, Martijn EG, van der Molen M, Schermer JG, Muskiet FAJ: Beta-N-terminal glycohemoglobins in subjects with common hemoglobinopathies: relation with fructosamine and mean erythrocyte age. Clin Chem 39:2259-65, 1993.
5Stamatoyannopoulos G, Nienhuis AW, Majerus PW, Varmus H: The Molecular Basis of Blood Diseases 2nd ed. W.B. Saunders, Philadelphia, 1994.
6Koskinen LK, Lahtela JT, Koivula TA: Fetal hemoglobin in diabetic patients. Diabetes Care 18:828-31, 1994.
Daniel E. Sabath, MD, PhD, is an associate professor of laboratory medicine (Head, Division of Hematology) and medicine (Division of Medical Genetics) at the University of Washington School of Medicine in Seattle.
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