Hemoglobin A1c Testing

Hemoglobin A1c: Hemoglobin is composed of a tetramer of globin chains; two of the chains are alpha-globin (chromosome 16). Most adults have hemoglobin that is largely comprised of two alpha chains combined with two beta chains (chromosome 11) (alpha2beta2), called HbA. The exons that encode beta-globin are adjacent to those encoding delta-globin and gamma-globin. About 2% of most adults’ hemoglobin is a variant (alpha2delta2), called HbA2. Less than 1% of most adults’ hemoglobin in fetal hemoglobin (alpha2gamma2), called HbF. A portion of HbA becomes glycosylated during its formation; a stable variety of glycosylated hemoglobin is called HbA1c.

HbA1c determinations are usually essential for determining a diabetic patient’s overall blood glucose control for the preceding 2-3 months; about one-half the value represents the prior 4 weeks. Diabetic complications of neuropathy, retinopathy, and nephropathy are unlikely to develop in a patient’s HbA1c remains consistently <7%. The recommended goals for HbA1c are either <7% (ADA) or <6.5% (ACE). Methods used to measure HbA1c include electrophoresis, boronate-affinity chromatography, immunoassay (eg, Bayer DCA-2000), and cation-exchange high pressure liquid chromotography (HPLC, eg: Bio-Rad Variant II).

Alpha thallasemia is due to a gene deletion causing reduced alpha-globin synthesis; since all hemoglobins contain alpha-globin, the percentage of HbF is not increased. Beta thallasemia is caused by point mutations causing reduced (?+) or absent (?0) beta-globin synthesis. The reduced beta-globin synthesis results in a relatively increased synthesis of delta-globins and gamma-globins, resulting in more HbA2 and HbF. In the presence of relatively excessive alpha-globin chains, the excessive alpha globin precipitates inside the RBC, damaging the RBC membrane and causing hemolysis. Patients who are heterozygous for a mutation in the beta-globin gene have beta thallasemia trait, also known as thallasemia minor. It is manifested by a lowish HCT/HGB and low RBCs.

HbF is usually elevated on a genetic basis, i.e., hereditary persistence of fetal hemoglobin (HPFH). HPFH can be due to either deletion of the delta-globin and beta-globin genes on the affected chromosome 11 or to point mutations in the promoter of one of the gamma-globin genes. Patients with beta thallasemia have increased production of HbF, as noted above; they can also co-inherit HPFH. (When inherited with sickle-cell anemia or beta-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.

The presence of increased amounts of HbF causes an underestmation of HbA1c by immunoassay. For example, the Bayer DCA-2000 immunoassay employs an antibody that specifically identifies the glycated amino terminus of only beta chains. In thallasemia minor, about 20% of hemoglobin is HbF that does not have beta chains; the assay does not detect the glycosylated gamma chains of HbF, resulting in a significant underestimation of glycosylated hemoglobin that may mislead the physician into thinking that the diabetes is in good control.

In contrast to the immunoassay, the HPLC method determines HbA1c levels by comparing the areas of the HbA1c and HbA peaks in the HPLC chromatogram. Because the glycated form of HbF migrates like HbA1c, the assay is not affected by the presence of high levels of HbF. Thus, HPLC is the most accurate method to measure HbA1c levels in patients with high HbF.

Other common hemoglobin variants, including Hb S, C, and E, do not interfere with either immunoassay or HPLC methods for HbA1c. They do not interfere with the immunoassay because the amino termini of the beta-globin chains of these variants are identical to that of the HbA beta-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 beta-globin chains are identical to those of HbA. Exceptions would include variant hemoglobins with mutations very close to the amino terminus of the beta-globin chain.

In contrast, hemoglobin variants that co-migrate with HbA1c on HPLC will interfere with HbA1c by HPLC, 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 life spans, so these patients will, on average, have lower HbA1c levels than expected for their actual level of glucose control. This is also true for patients who have short red cell life spans for other reasons, including hemolytic anemia.

Patients receiving HAART therapy for AIDS may have a hemoglobin abnormality, evidenced by increased MCV and MCHC. I have personally observed a misleadingly low HbA1c in a patient with diabetes type 1 who has AIDS due to HIV and is being treated with HAART.

One cannot know a priori whether a patient’s immunoassay HbA1c level is 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, particularly HbF. Because the National Glycohemoglobin Standardization Program now standardizes most HbA1c methods in use, the HbA1c levels determined by different HPLC methodologies should be comparable.

  • UCSF laboratory employs an HPLC assay for HbA1c, performed at China Basin.
  • Quest laboratory employs an “immunoturbigometric” assay for HbA1c.
  • LabCorp laboratory employs an immunoassay for HbA1c.

Sabatg DE: Case study: artifactually low hemoglobin A1c in a patient with high hemoglobin F. Clinical Diabetes 18: 2000. (Parts of this article were adapted for this discussion.)

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