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In the mid-to-late 1990s, results from the Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS) showed conclusively that intensive glycemic control significantly reduced the risk and progression of long-term complications of diabetes.1,2 These results highlighted the use of HbA1c for monitoring of glycemic control in diabetes and set the stage for the use of specific diabetes treatment goals using HbA1c. Since that time, most clinical diabetes organizations have included specific goals for diabetes treatment in terms of HbA1c. Since small differences in %HbA1c translate into large differences in the rate of progression of complications, it is extremely important for HbA1c results to be accurate and precise, with harmonization between methods and laboratories. The National Glycohemoglobin Standardization Program (NGSP), along with manufacturers, have improved the quality of HbA1c methods over the last 15–20 years, but there is still a need for even greater accuracy and precision with some available methods.

The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) provides a definitive reference method for HbA1c that facilitates traceability to a true accuracy base. There is a tight linear correlation between results of the NGSP and IFCC networks; however, the actual numbers reported by the NGSP and IFCC are different, which has led to controversy over which numbers should be reported by laboratories. In 2007 and again in 2010, consensus statements recommended the reporting of both numbers worldwide: NGSP in %HbA1c and IFCC in mmol/mol Hb. Importantly, the relationship between the NGSP and IFCC laboratory networks has been established in a “master equation”: NGSP% = (0.0915 × IFCC [mmol/mol] + 2.15), which continues to be monitored.3 The two groups now serve complementary purposes: the IFCC provides manufacturers with secondary reference materials with which to anchor their methods and the NGSP provides certification with defined acceptable performance limits that are based on clinical requirements. Many countries have already recommended the reporting of both numbers, but the USA will continue to report only the NGSP %HbA1c according to American Diabetes Association (ADA) recommendations.

Regardless of which number is reported (NGSP or IFCC), it is essential that HbA1c be measured with precision and accuracy to enable healthcare providers to reliably distinguish optimal from suboptimal glycemic control. The recent recommendation to use HbA1c for the diagnosis of diabetes further emphasizes the need for optimal assay performance.4,5

Current assay performance

  1. Top of page
  2. Current assay performance
  3. Are HbA1c results good enough for optimal use of the test?
  4. HbA1c interferences
  5. Summary
  6. References

In order to improve method performance for HbA1c, the NGSP and the College of American Pathologists (CAP) have been progressively tightening their acceptance limits for method certification and laboratory performance, respectively. HbA1c assay performance has improved considerably over the past 10 years, with overall coefficients of variation (CVs) (across different methods in different laboratories) now approximating 4%. In the normal range, the decrease has been from CVs of 7% down to <4% over the past 10 years.6 Between-laboratory CVs for some methods are <2%. Figure 1 shows the mean ± 2SD for each method on the 2010 survey compared to the CAP limits. Clearly some methods are better than others, but 95% of laboratories are reporting results within 8% of the NGSP target (CAP acceptance limit).7 This CAP limit will be tightened in 2011 to ±7% in order to motivate manufacturers and laboratories to improve further.

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Figure 1.  Mean and 2SD bars for each method group in the 2010 College of American Pathologists (CAP) GH2-B survey for the mid-level HbA1c sample. The dotted line shows the National Glycohemoglobin Standardization Program (NGSP) target value and the shaded area indicates the ±8% acceptable CAP limit.

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Are HbA1c results good enough for optimal use of the test?

  1. Top of page
  2. Current assay performance
  3. Are HbA1c results good enough for optimal use of the test?
  4. HbA1c interferences
  5. Summary
  6. References

Many people believe that performance goals for HbA1c testing should be based on clinical requirements. There are two important questions for a physician to consider when using HbA1c measurements to assess a patient’s glycemic status. One question is whether the patient’s glycemic control is stable, improving or deteriorating, and the second is how the HbA1c result compares to the individual’s target HbA1c (the current ADA recommendation is 7% with the qualification that lower is better if feasible). In answer to the first question, many physicians have suggested that 0.5% HbA1c is a “clinically significant change”. Importantly, treatment guidelines and algorithms from various clinical organizations recommend evaluating new treatment regimens in terms of whether HbA1c is lowered by 0.5% points or more.8,9 Therefore, it is important to be sure that a change of this magnitude is statistically significant and not due to analytical variation. Taking a statistically significant difference of 0.5% HbA1c at a HbA1c concentration of 7% as our goal for HbA1c measurement, one can use the reference change value (RCV), to calculate an appropriate goal in terms of a method’s CV.10 For sequential results to be significantly different, the numbers must differ by more than the combined variation inherent in the two results. Assuming a within-subject biological variation of <1%,11 if the analytical CV of a HbA1c method is 2% (feasible for many commercially available HPLC systems), then the RCV (95% probability) is <0.5% HbA1c (0.43%).

We can conclude that, at least when HbA1c is measured in an accredited laboratory with an accurate and precise assay method, physicians can be reasonably (95%) certain that a difference of ≥0.5% HbA1c between successive patient samples represents a statistically significant change in glycemic control. If the laboratory is using a method that demonstrates a within-laboratory CV of >2%, as is still the case with some assay methods, there is less confidence that the difference of 0.5% HbA1c is significant.

In the situation where a physician wants to look at the difference between a patient’s result and a goal of 7% HbA1c, both the bias and variability (% CV), in other words the total error, of the method must be taken into account. For example, if a method has zero bias, a CV of 3.5% is required to have 95% confidence that the HbA1c result for a patient with a “true” result of 7% will read between 6.5% and 7.5% (±7%). If there is a bias of 0.2% HbA1c, the CV requirement would tighten to 2.3%. While we have made considerable progress in improving HbA1c method performance, it is clear that further improvement is necessary in order for the test to be used optimally. Hopefully, further tightening of NGSP and CAP limits will lead to better accuracy and precision of HbA1c methods.

HbA1c interferences

  1. Top of page
  2. Current assay performance
  3. Are HbA1c results good enough for optimal use of the test?
  4. HbA1c interferences
  5. Summary
  6. References

Another area of concern, other than precision and accuracy, is the potential for erroneous HbA1c results due to interference. HbA1c can be accurately assessed in the vast majority of subjects, but in cases where the subject may have a medical condition that can interfere with HbA1c, or if a hemoglobin variant may be present, the results should be interpreted with caution. Conditions such as hemolytic anemia, renal failure or polycythemia can alter the red cell lifespan, thereby falsely lowering or increasing HbA1c results. Severe iron-deficiency anemia has also been reported to interfere with HbA1c results to some degree.12

The effects of common hemoglobin variants on HbA1c results have been studied extensively13–15 and these findings are summarized on the NGSP web site.16 Hemoglobin variants usually occur in the heterozygous form; for these individuals and individuals with elevated HbF interferences are method-specific and HbA1c can be measured accurately if an appropriate assay method is used. HbAS and HbAC (two of the most common variants in the world) interfere with some immunoassays, although the most widely used immunoassays are now free of this interference. HbAE and HbAD interfere with some HPLC methods, but do not affect immunoassays. With ion-exchange methods, chromatograms generally show aberrant peaks, indicating the presence of a variant and thus enabling detection of unacceptable HbA1c results prior to their being reported. For subjects with HbF >10%, a method that does not show interference from this variant should be used. As with any test, results that are inconsistent with the clinical presentation should be investigated further. For individuals with certain homozygous or double heterozygous variants (e.g. HbSS, HbCC, and HbSC) that cause a shortened red cell lifespan, the HbA1c results will be inaccurate regardless of the assay method, and alternative methods for assessing glycemic control must be used.

Other factors have been reported to influence HbA1c results. Reports have indicated that HbA1c increases slightly with age, even after adjusting for blood glucose levels,17 but there is some question as to whether this necessitates age-specific treatment goals. It has also been reported that HbA1c results vary slightly among ethnic groups,18,19 but there is some controversy over the clinical significance of these differences and whether they are due to differences in mean blood glucose or other factors.

Summary

  1. Top of page
  2. Current assay performance
  3. Are HbA1c results good enough for optimal use of the test?
  4. HbA1c interferences
  5. Summary
  6. References

The NGSP has been highly successful in terms of reducing the variability of HbA1c results among methods and laboratories by harmonizing results to clinical trials that established the relationships between HbA1c and the risks for diabetic complications. The IFCC reference method and materials facilitate metrological traceability of HbA1c results to a higher order reference system. The issue of which numbers are to be reported in clinical settings (NGSP% or IFCC mmol/mol Hb) is being decided on a country-by-country basis, but there is an established equation where results can easily be converted between the two systems.

Nonetheless, there is still room for improvement. While many assay methods demonstrate performance that is sufficient to meet clinical needs (in terms of reliably distinguishing differences of 0.5% HbA1c), some still do not. Moreover, optimal assay performance is more crucial than ever, given the recent recommendation to use HbA1c for the diagnosis of diabetes. Future reductions in the NGSP and CAP acceptance limits are likely, and the impact of these measures on the quality of HbA1c results will continue to be evaluated. Additionally, the NGSP will continue to evaluate the impact of potential interferences on HbA1c results, with the goal of encouraging the development and use of methods that are free of common interferences. Taken together, these measures will hopefully ensure that HbA1c testing is sufficiently reliable to meet clinical needs.

References

  1. Top of page
  2. Current assay performance
  3. Are HbA1c results good enough for optimal use of the test?
  4. HbA1c interferences
  5. Summary
  6. References
  • 1
    DCCT Research Group. The effect of intensive treatment of diabetes on the development and progression of long term complications in insulin dependent diabetes mellitus. N Engl J Med. 1993; 329: 97786.
  • 2
    UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet. 1998; 352: 83751.
  • 3
    Geistanger A, Arends S, Berding C et al. Statistical methods for monitoring the relationship between the IFCC reference measurement procedure for hemoglobin A1c and the designated comparison methods in the United States, Japan, and Sweden. Clin Chem. 2008; 54: 137985.
  • 4
    International Expert Committee. International Expert Committee report on the role of the A1c assay in the diagnosis of diabetes. [see comment]. Diabetes Care. 2009; 32: 132734.
  • 5
    American Diabetes Association. Standards of medical care in diabetes. Diabetes Care. 2010; 33: S1161.
  • 6
    Little RR, Rohlfing CL, Sacks DB, for the National Glycohemoglobin Standardization Program Steering C. Status of hemoglobin A1c measurement and goals for improvement: from Chaos to order for improving diabetes care. Clin Chem. DOI: 2010.148841.
  • 7
    College of American Pathologists. Surveys 2010 GH2-A Glycohemoglobin Participant Summary. Vol. College of American Pathologists, Nothfield (IL), 2010.
  • 8
    Nathan DM, Buse JB, Davidson MB et al. Medical management of hyperglycemia in type 2 diabetes: a consensus algorithm for the initiation and adjustment of therapy: a consensus statement of the American Diabetes Association and the European Association for the Study of Diabetes. Diabetes Care. 2009; 32: 193203.
  • 9
    National Institute for Health and Clinical Excellence (NICE). Type 2 diabetes: newer agents for blood glucose control in type 2 diabetes. Available from: http://www.nice.org.uk/nicemedia/live/12165/44318/44318.pdf (Accessed September 2010).
  • 10
    Fraser CG, Harris EK. Generation and application of data on biological variation in clinical chemistry. Crit Rev Clin Lab Sci. 1989; 27: 40937.
  • 11
    Rohlfing C, Wiedmeyer HM, Little R et al. Biological variation of glycohemoglobin. Clin Chem. 2002; 48: 11168.
  • 12
    Tarim O, Kucukerdogan A, Gunay U, Eralp O, Ercan I. Effects of iron deficiency anemia on hemoglobin A1c in type 1 diabetes mellitus. Pediatr Int. 1999; 41: 35762.
  • 13
    Little RR, Rohlfing CL, Hanson S et al. Effects of hemoglobin (Hb) E and HbD traits on measurements of glycated Hb (HbA1c) by 23 methods. Clin Chem. 2008; 54: 127782.
  • 14
    Mongia SK, Little RR, Rohlfing CL et al. Effects of hemoglobin C and S traits on the results of 14 commercial glycated hemoglobin assays. Am J Clin Pathol. 2008; 130: 13640.
  • 15
    Rohlfing CL, Connolly SM, England JD et al. The effect of elevated fetal hemoglobin on hemoglobin A1c results: five common hemoglobin A1c methods compared with the IFCC reference method. Am J Clin Pathol. 2008; 129: 8114.
  • 16
    NGSP. HbA1c assay interferences. Available from: http://www.ngsp.org/interf.asp (accessed November, 2010).
  • 17
    Pani LN, Korenda L, Meigs JB et al. Effect of aging on A1c levels in individuals without diabetes: evidence from the Framingham Offspring Study and the National Health and Nutrition Examination Survey 2001–2004. Diabetes Care. 2008; 31: 19916.
  • 18
    Herman WH, Ma Y, Uwaifo G et al. Differences in A1c by race and ethnicity among patients with impaired glucose tolerance in the Diabetes Prevention Program. Diabetes Care. 2007; 30: 24537.
  • 19
    Herman WH, Dungan KM, Wolffenbuttel BH et al. Racial and ethnic differences in mean plasma glucose, hemoglobin A1c, and 1,5-anhydroglucitol in over 2000 patients with type 2 diabetes. J Clin Endocrinol Metab. 2009; 94: 168994.