Case 1

A 55-year-old man of Southeast Asian descent presented with abnormal renal function for investigation. He had a 6-month history of gradual but progressive lethargy, tiredness, and poor concentration. There was no history of inherited or acquired kidney disease, and he had been fit and healthy before recent onset of symptoms. He had a strong family history of type 2 diabetes with an affected father, aunt, and brother. He was a nonsmoker and consumed ∼ 0.35 oz of alcohol daily.

Clinically, he appeared well, with a blood pressure of 160/105 mmHg and regular pulse of 88 bpm. His weight was 222 lb (110 kg) and height 5′8″ (1.72 m) (BMI ∼37 kg/m2). Fundoscopy showed evidence of early nonproliferative retinal disease with macula sparing. His general examination was otherwise normal, with no evidence of nephromegaly,renal bruit, or microvascular disease.

Urinalysis revealed the presence of proteinuria, confirmed to be 1.8 g/day with no hematuria. He had a blood urea nitrogen (BUN) level of 99.7 mg/dl and creatinine of 6.1 mg/dl with sodium of 136 mmol/l and potassium of 5.4 mmol/l. His fasting glucose level was 122.5 mg/dl.

A follow-up oral glucose tolerance test (OGTT) confirmed the presence of impaired fasting glycemia and impaired glucose tolerance, with a fasting glucose level of 117.1 mg/dl (6.5 mmol/l) and a 2-hour post-OGTT glucose level of 160.4 mg/dl (8.9 mmol/l). His hemoglobin A1c (A1C) levels(performed by high-performance liquid chromatography) were 4.5 and 4.2% on two separate occasions with abnormal chromatograms(Figure 1). Follow-up electrophoresis revealed the presence of hemoglobin E. Further investigation of his renal abnormality, including biopsy, confirmed the presence of diabetic nephropathy, with no other causes for chronic renal failure being apparent. On follow-up, his diabetes control was excellent with dietary and lifestyle management.

Figure 1.

E pattern hemoglobinopathy interfering with the analysis of A1C value.

Figure 1.

E pattern hemoglobinopathy interfering with the analysis of A1C value.

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Case 2

A 60-year-old woman with long-standing obesity and hypertension presented concerned about the possibility of type 2 diabetes given her strong family history of the disease. Clinical examination was unremarkable, with a blood pressure of 150/80 mmHg. Her fasting glucose was 117.1 mg/dl (6.5 mmol/l) and A1C on two separate occasions was 5.5 and 4.5%. The patient was reassured that she did not have diabetes and was discharged from the clinic without follow-up.

One year later, her fasting glucose was 122.5 mg/dl (6.8 mmol/l). A follow-up OGTT revealed a fasting glucose of 124.3 mg/dl (6.9 mmol/l) and a 2-hour glucose of 214.4 mg/dl (11.9 mmol/l), diagnostic of diabetes. Her A1C was measured again and found to be 5.3%. In contrast to her low A1C, her serum fructosamine was elevated at 312 μmol/l, consistent with the presence of chronic hyperglycemia.

A review of her hemoglobin chromatogram(Figure 2) showed an abnormal peak interfering with the isolation of the glycated hemoglobin. This was confirmed with the repeat measurement. Her hemoglobin electrophoresis study showed an abnormal hemoglobin component. This was further confirmed to be hemoglobin British Columbia.

Figure 2.

Abnormal peak of hemoglobin British Columbia on the hemoglobin chromatogram.

Figure 2.

Abnormal peak of hemoglobin British Columbia on the hemoglobin chromatogram.

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  1. What is A1C, and how relevant is it to the control of diabetes?

  2. What are the potential confounders of A1C use in the assessment of patients with diabetes?

  3. What are the available alternatives to A1C as markers of glycemic control?

  4. Are A1C values diagnostic of diabetes?

A1C is the nonenzymatic glycated product of the hemoglobin beta-chain at the valine terminal residue. The number 1c following HbA represents the order in which this hemoglobin is detected on chromatography. Hence, other hemoglobin peaks are referred to as HbA1a1, HbA1a2,HbA1b, and so forth.

The A1C constitutes about 60-80% of total glycated hemoglobin. It is normally present, albeit at low levels, in circulating red cells because of the glycosylation reaction between hemoglobin and circulating glucose.1  In the presence of excess plasma glucose, the hemoglobin beta-chain becomes increasingly glycosylated, making the A1C a useful index of glycemic control. The importance of A1C as an index of diabetes control was reinforced by the Diabetes Control and Complications Trial(DCCT).2  This study demonstrated a direct correlation between glycemic control as indicated by A1C and the likelihood of developing long-term diabetes-related complications.

Because A1C is based on hemoglobin, both qualitative and quantitative variations in hemoglobin can affect the A1C value. These factors need to be considered when interpreting A1C results and serve to limit the use of A1C as a diagnostic test for diabetes.

Clinicians should also appreciate the differences in assay methods for A1C,which have relevance to the possibility of interference. In the case of reduced total hemoglobin or increased turnover of red blood cells (RBCs), the level of A1C will be reduced even in the presence of high ambient plasma glucose, thereby falsely lowering the A1C and limiting its usefulness as a measure of glycemia. In situations where the A1C is low, contrary to high day-to-day glucose levels, attention should be paid to the hemoglobin concentration, the blood smear, and possibly hemolytic parameters to rule out the presence of anemia or hemolysis.

The other pathophysiological process that can affect the A1C value is the structure of hemoglobin itself. Qualitatively, any disorder that affects hemoglobin production, particularly the beta-chain, will affect the A1C results. In the case of patients with beta-thalassemia, the absence of beta-hemoglobin chains for glycosylation invalidates the use of A1C. In other hemoglobinopathies, there is often the combination of abnormal hemoglobin plus associated excessive intramedullary hemolysis. These, in turn, will lead to a falsely low A1C.

Case 1 illustrates this well. The abnormality in hemoglobin E is a point substitution of glutamine for lysine at position 26 on the beta-chain (B26 glu→ lys). Patients with this kind of hemoglobinopathy are likely to form glycated hemoglobin E1c instead of A1C, leading to a low A1C level.3  In Case 2,the abnormality in the hemoglobin British Columbia was found to be at codon number 101 [Glu (GAG) → Lys (AAG)] on the beta-chain, which interferes with glycosylation and hence falsely lowers the A1C level.4  Any suspicion of a discordant A1C level should be followed up with a review of the hemoglobin chromatogram for any abnormal peaks and hemoglobin electrophoresis,if indicated.

The immunoassay technique used is another potential interference with the measurement of A1C. This method employs various antibodies to detect the A1C fraction. If the antibodies recognize specifically the N-terminus of the beta-chain, this assay will deliver falsely low results in situations where the number of beta-chains is either abnormal or reduced as demonstrated in our two cases. If not suspected, patients may be thought to have better glycemic control than is actually the case due to falsely low A1C results.

In the presence of renal failure, the clinical utility of A1C is even more questionable. The hemoglobin in renal disease gets carbamylated due to condensation of urea-derived cyanate with the N-terminal amino groups. This can subsequently read as a high A1C result when detected by common methods such as ion-exchange chromatography. The A1C level in renal failure thus represents a balance between the anemia associated with renal disease and hemoglobin adducts in renal failure. These two factors often balance each other out with the eventual outcome being that the A1C value is unchanged. Therefore, A1C could be employed usefully as a marker for diabetes-related complications in patients with uremia.5  The combination of anemia and hemoglobinopathy resulted in a falsely low A1C level in Case 1.

Other useful markers for diabetes control include total glycohemoglobin,which does not take into account the hemoglobin beta-chain and other blood-based glycated proteins such as fructosamine. The latter is also readily available in most laboratories and is reflective of mean glycemia but over a shorter time of 15-30 days compared with 60-120 days of A1C.6  While useful,these tests have not been as well validated as A1C. Although they have not been proven to reliably predict diabetes complications, extrapolation of the DCCT data would suggest they should also be useful for this purpose. Otherwise, in the presence of abnormal hemoglobin, one is left with day-to-day variations in blood glucose readings with which to monitor glycemic control.

Despite previous reports7,8 advocating it, the use of A1C as a tool for the diagnosis of diabetes is at best controversial.8-10 The American Diabetes Association does not recommend its use as a diagnostic tool and suggests it should only be used for monitoring diabetes.11  The Australian and New Zealand position statement regarding new classification and criteria for diagnosis of diabetes makes no reference to the use of A1C.12  Although it can be falsely low, there are other conditions that can lead to a falsely elevated A1C, including alcoholism,13  lead poisoning, opiate addiction,1 excessive use of salicylate (due to interference),3  and pregnancy. The increase is often small and is not of clinical relevance,leaving diabetes in the majority of cases as the primary cause of A1C elevation.

  • A1C is an important marker of glycemic control in patients with diabetes.

  • A1C is subjected to interference in the presence of associated comorbidities including hemoglobinopathies, hemolysis, renal failure, and alcoholism.

  • Its use in the diagnosis of diabetes is controversial and not recommended.

Huy A. Tran, FACE, FRACP, FRCPA, is director of the Division of Clinical Chemistry of the Hunter Area Pathology Service at John Hunter Hospital in Newcastle, Australia. Diego Silva, MD, is a research fellow at the Autoimmune Research Unit of the Australian National University in Canberra. Nikolai Petrovsky, FRACP, PhD, is a professor and director of the National Health Sciences Unit of the Australian National University and a senior endocrinologist in the Department of Endocrinology and Diabetes of the Canberra Hospital in Canberra.

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