Focus: HbA1c and postprandial glucose: should they be used as diagnostic criteria or only for monitoring glycemic control?






Giorgio SESTI,MD
Department of Medical and Surgical Sciences
University Magna Græcia of Catanzaro – ITALY

HbA1c and postprandial glucose: should they be used as diagnostic criteria or only for monitoring glycemic control?


by G. Sesti, Italy



In 2009, an International Expert Committee comprising representatives of the International Diabetes Federation, the American Diabetes Association (ADA), and the European Association for the Study of Diabetes recommended measurement of glycated hemoglobin (HbA1c) as a diagnostic test option for diabetes (with a threshold of ≥6.5%) in addition to using glucose criteria. In 2010, the ADA adopted this criterion for the diagnosis of diabetes, and revised criteria for prediabetes diagnosis (impaired fasting glucose and impaired glucose tolerance), recommending that HbA1c values of 5.7% to 6.4% be used as an indicator of prediabetes. HbA1c testing offers some advantages, including lower day-to-day variability, less perturbation during periods of stress or illness, and requirement of a nonfasting sample. However, diagnosis based on HbA1c may identify individuals with type 2 diabetes or prediabetes that are distinct from those identified based on glucose criteria. The final goal would be to have a single universal diagnostic test for diabetes. Unfortunately, an increasing amount of evidence suggests that ethnic differences in HbA1c values may be present, and some clinical situations, such as anemia and hemoglobinopathies, interfere with accurate HbA1c measurement. Given the higher costs, which are unaffordable in many countries, the recommendation to use HbA1c as the only diagnostic test for diabetes on a global scale is not possible at present, and the glucose assay will continue to have an important role in the diagnosis of diabetes and prediabetes.

Medicographia. 2013;35:95-101 (see French abstract on page 101)



Glycated hemoglobin (HbA1c) is an integrated measure of average blood glucose levels over the preceding 2-3 months and is broadly used for monitoring metabolic control in individuals with diabetes.1 In 2009, an International Expert Committee comprising representatives of the International Diabetes Federation (IDF), the American Diabetes Association (ADA), and the European Association for the Study of Diabetes (EASD) recommended the use of HbA1c measurement as a diagnostic test option for diabetes (with a threshold of ≥6.5%) in addition to using criteria based on either fasting plasma glucose (FPG) or the 2-h 75-g oral glucose tolerance test (OGTT): an FPG level ≥126 mg/dL and 2-h post load plasma glucose (2-h PG) ≥200 mg/dL, respectively (Table I, page 96).2,3

This decision was supported, at least in part, by epidemiological studies showing a similar relationship between HbA1c and risk of retinopathy as has been observed for FPG and 2-h PG concentrations. In 2010, the ADA adopted this criterion for the diagnosis of diabetes, and revised criteria for identifying those within a category of increased risk for diabetes (prediabetes), which includes those with impaired fasting glucose (IFG) and impaired glucose tolerance (IGT).3 Specifically for the categories of increased risk for type 2 diabetes, the ADA recommendations state that an HbA1c value from 5.7% to 6.4% identifies persons at increased risk for diabetes (Table II).3 It is important to note that the HbA1c assay should be performed using a method that is certified by the National Glycohemoglobin Standardization Program (NGSP) and standardized to the Diabetes Control and Complications Trial (DCCT) reference assay, whereas point-of-care HbA1c measurement is not sufficiently accurate at present to use for diagnostic aims.


Table I
Table I. Criteria for the diagnosis of diabetes.

Abbreviations: DCCT, Diabetes Control and Complications Trial; HbA1c, glycated
hemoglobin; NGSP, National Glycohemoglobin Standardization Program; OGTT,
oral glucose tolerance test.
After reference 3: American Diabetes Association. Diabetes Care. 2012;35
(suppl 1):S64-S71. © 2012, American Diabetes Association.



Table II
Table II. Categories of increased risk for diabetes (prediabetes).

Abbreviations: FPG, fasting plasma glucose; HbA1c, glycated hemoglobin;
OGTT, oral glucose tolerance test.
After reference 3: American Diabetes Association. Diabetes Care. 2012;35
(suppl 1):S64-S71. © 2012, American Diabetes Association.



Although the glucose assay is considered the “gold standard” for diagnosis of diabetes, blood glucose measurement has many limitations, several of which are not well recognized. HbA1c measurement, although appealing, has some intrinsic limitations as well. This article will provide an overview both of the factors influencing glucose and HbA1c testing and of the pros and cons of using HbA1c to diagnose diabetes.

Factors contributing to variation in the glucose assay

_ Fasting plasma glucose
FPG is the most commonly accepted diagnostic criterion for diabetes because the assay is available in most laboratories worldwide, inexpensive, easily automated, and requires a single sample. Nevertheless, fasting blood sampling interferes with daily activities, such as work, increasing the possibility that a person with type 2 diabetes will remain undiagnosed. Furthermore, FPG assessment has some limitations, including low reproducibility. For example, in a study from the Third National Health and Nutrition Examination Survey (NHANES III) that analyzed repeated assays from 685 fasting participants without diagnosed diabetes, only 70.4% of individuals with FPG ≥126 mg/dL on the first test were confirmed as having FPG ≥126 mg/dL when the analysis was repeated 2 weeks later.4 Several factors may contribute to this low reproducibility. FPG concentrations vary noticeably both in a single person from day to day (intraindividual variation in a healthy person is reported to be 5.7%-8.3%) and also between two or more people (interindividual variation in a healthy person is reported to be up to 12.5%). Thus, based on a coefficient of variation (CV) of 5.7%, FPG can range from 112-140 mg/dL in a person with an FPG of 126 mg/dL. Other factors may affect blood test results such as medications, venous stasis, posture, and sample handling. Plasma glucose concentration can be altered by prolonged fasting, physical activity, a hypocaloric diet for a week or more prior to testing, or food ingestion. Intercurrent illness, and acute stress can alter blood glucose concentrations.5 FPG has also a diurnal variation. A study of 12 882 participants in NHANES III who had no previously diagnosed diabetes revealed that mean FPG in the morning is considerably higher than in the afternoon.6

Accuracy is another problem in basing diabetes diagnosis on the plasma glucose assay. Glucose concentrations decrease in the test tube by 5%-7% per hour as a consequence of glycolysis.7 To provide a correct assay of plasma glucose, the sample should be placed on ice water immediately and spun to isolate the plasma within 60 min. However, this is not usual procedure followed in clinical practice. If the sample is left unprocessed at room temperature, the cells in the blood will continue to consume glucose, and consequently a sample with a true blood glucose concentration of 126 mg/dL would have a glucose concentration of about 110 mg/dL after 2 h. Rates of glycolysis are even higher in samples with increased concentrations of erythrocytes, white blood cells, or platelets.





The nature of the sample analyzed has a considerable influence on the glucose concentration assay. Glucose can be measured in whole blood, plasma, or serum, but plasma is recommended by both the ADA and World Health Organization (WHO) for diagnosis of diabetes.2,8 Nonetheless, numerous laboratories measure glucose in serum, and these values may differ from those obtained by measuring glucose in plasma. Glucose concentrations in whole blood are 11% lower than those in plasma because erythrocytes have a lower water content than plasma, depending also on hematocrit value.7 The majority of glucometers use whole blood for measuring glucose in capillary blood, but the results are not accurate in patients with anemia unless the meter measures hematocrit.9 In addition, capillary glucose concentrations can be 20%-25% higher than venous glucose during an OGTT,10 and thus is unpractical for the OGTT; however, capillary sampling is widely used, particularly in underresourced countries, and WHO considers capillary blood samples suitable for the diagnosis of diabetes.8

Glucose is measured in almost all laboratories using enzymatic methods, predominantly with glucose oxidase and with good precision (CV<2.5%). However, there is no program to standardize results among different automated instruments and different laboratories. In a study comparing serum glucose measurements performed in 6000 laboratories using 32 different instruments, a statistically significant difference in bias (deviation of the result from the true value) has been observed among laboratories, with biases ranging from –6 to +7 mg/dL at a glucose concentration of 100 mg/dL.11 These differences among laboratories can result in the potential misclassification of up to 12% of patients.2

_ Oral glucose tolerance test
One in three cases of undiagnosed type 2 diabetes in Europe will have nondiabetic fasting values, and therefore an OGTT will be required for diagnosis. The OGTT assesses the efficiency of the body to metabolize a standard dose of glucose (75 g) ingested orally. FPG and 2-h PG reflect different aspects of glucose metabolism. A combination of hepatic insulin resistance and defective early-phase insulin secretion resulting in excessive fasting hepatic glucose production contributes to fasting hyperglycemia. By contrast, a near-normal hepatic insulin sensitivity and marked muscle insulin resistance combined with defective late insulin secretion contribute to hyperglycemia after a glucose load.12 A rise in postprandial glucose concentration frequently occurs before fasting glucose increases. Therefore, postprandial glucose is a sensitive indicator of the risk for developing diabetes and an early marker of impaired glucose metabolism. In addition, compelling evidence suggests that elevated 2-h PG level during an OGTT is a better predictor of cardiovascular mortality or morbidity than FPG.13,14 However, the use of the OGTT for diagnosis of diabetes has several disadvantages. The OGTT is time-consuming and tiresome for the person, expensive, and influenced by numerous medications and conditions other than diabetes; the dose of ingested glucose is unpalatable, and extensive subject preparation is required including ingestion of at least 150 g of dietary carbohydrate per day for 3 days prior to the test, a 10- to 16-h fast, and commencement of the test between 7:00 AM and 9:00 AM. In addition, the OGTT is subject to the same limitations as the FPG assay, as above described. Finally, a high degree of intraindividual variability in the OGTT, with a CV of 16.7%, considerably greater than the variability for FPG, has been reported,4 resulting in poor reproducibility of the OGTT.15,16 For these reasons, the ADA recommends FPG measurement as the preferred glucose based diagnostic test.3

Factors contributing to variation in the HbA1c assay

HbA1c is formed by glycation of the NH2-terminal valine residue of the β chain of globin. The average life span of erythrocytes is approximately 120 days and, therefore, HbA1c concentrations reflect the average glycemic exposure over the preceding 2-3 months. Chronic hyperglycemia is strongly associated with microvascular complications of diabetes. Observational studies show a strong correlation between HbA1c and such complications.17-19 In one study in which both FPG and HbA1c were measured, there was a stronger correlation between HbA1c and retinopathy than between FPG levels and retinopathy.18 Importantly, lowering HbA1c concentrations by tight glycemic control significantly reduces the rate of progression of microvascular complications, in particular retinopathy,20-24 and these findings have been used to establish the HbA1c treatment target for diabetes care.3 Taken together, these observations suggest that a reliable measure of glucose concentrations, such as HbA1c, which captures long-term glycemic exposure and is related to the risk of diabetes-specific complications, may serve as a better biochemical marker for the occurrence and severity of the disease than single or episodic measures of glucose levels.

HbA1c measurements offer some practical advantages over assessments of FPG or glucose levels during an OGTT. HbA1c levels have lower day-to-day variability, with a CV of <2%;25 they are not affected by recent food ingestion, intense exercise, stress, or illness, and samples can be taken at any time of the day and are stable for 1 week at 4°C; furthermore, concentrations predict the development of microvascular diabetes complications, and are used to monitor treatment. It is important to note that the diagnostic test should be performed by a method that is certified by the NGSP and standardized to the DCCT reference assay. Unfortunately, international standardization of the HbA1c assay has not yet been fully realized, and point-of-care HbA1c assays are not sufficiently accurate at this time to be recommended for diagnostic aims. Screening for diabetes by measuring the HbA1c concentrations rather than glucose levels is more expensive and, therefore, unaffordable in many low- and middle-income countries.

In addition, interindividual variation is greater, with HbA1c concentrations varying among individuals despite the presence of similar blood glucose levels.26 There is an increasing amount of evidence suggesting that race influences HbA1c. HbA1c concentrations have been reported to be higher among Hispanics, African Americans, American Indians, and Asians than in whites before and after adjusting for several confounders such as age, gender, body mass index, duration of diabetes, oral medication use, mean FPG, mean postprandial glucose levels, insulin resistance, andβ-cell function.27-29 However, the variations in HbA1c levels are relatively small (0.4%-0.7%), and there is no consensus on whether different cut points should be used for different ethnic groups. The molecular mechanisms underlying these disparities remain unsettled. Differences in intraerythrocyte 2,3-diphosphoglycerate, which catalyzes production of HbA1c, with subsequent impact on rates of glucose transport into erythrocytes, rates of intraerythrocytic glucose metabolism, rates of glucose binding to or release from hemoglobin, or erythrocyte life span might account for these ethnic disparities.30,31

Interpretation of HbA1c measurements is influenced by the erythrocyte life span. Individuals with anemia, hemolytic disease, or other conditions with abnormal turnover of red cells, such as pregnancy, malaria, and recent blood loss or transfusion have a substantial reduction in HbA1c levels.32 For these conditions, the diagnosis of diabetes should be based exclusively on glucose criteria. Spurious increases in HbA1c levels have been reported with some methods in subjects with uremia, hyperbilirubinemia, hypertriglyceridemia, chronic alcoholism, or chronic ingestion of salicylates. A recent study comparing the diagnostic performance of HbA1c against a standard OGTT in young Indian adults showed an unexpectedly high prevalence of prediabetes and diabetes based on HbA1c measurement (25.9%), as compared with prevalence based on the OGTT (10.4%).33 Hematological parameters that explained 13.1% of the variance in HbA1c concentrations included anemia and erythrocyte indices indicative of iron deficiency, suggesting that the use of HbA1c to diagnose prediabetes and diabetes in iron-deficient populations may lead to a spuriously exaggerated prevalence. A potential mechanism explaining the association between iron deficiency and higher HbA1c levels has been identified by the demonstration that malondialdehyde, which is increased in individuals with iron deficiency anemia,34 increases glycation of globin.35

Abnormal hemoglobin traits, such as HbS, HbC, HbF, HbD, and HbE affect some HbA1c measurements. The HbA1c assay is not appropriate in homozygous carriers of HbS or HbC, those with HbSC, or with any other variant that affects erythrocyte survival. Nonetheless, HbA1c can be measured accurately in heterozygous carriers of HbS, HbE, HbC, or HbD, and in individuals with increased HbF, provided an appropriate assay is used (an updated list is available at www.ngsp.org/ npsp.org/interf.asp). Despite these caveats, HbA1c can be measured accurately in the majority of people.

Comparison of HbA1c, FPG, and 2-h PG criteria to diagnose diabetes and prediabetes

Although it would be desirable for FPG, 2-h PG, and HbA1c values to be equivalent in identifying persons with diabetes or prediabetes, poor concordance between the three diagnostic criteria has been reported in different ethnic groups.36-40 We compared HbA1c, FPG, and 2-h PG criteria for the diagnosis of diabetes in a cohort of white Italians comprising 1019 individuals without known diabetes.40 Moderate agreement existed for HbA1c and FPG criteria for diagnosis of type 2 diabetes (κ coefficient = 0.522), with 85.5% of subjects classified as not having diabetes by both HbA1c and FPG criteria, and 5.8% classified as having diabetes by both HbA1c and FPG criteria. Discordant classifications occurred for 5.5% of subjects with HbA1c ≥6.5% and FPG <126 mg/dL and for 3.2% of subjects with HbA1c <6.5% and FPG ≥126 mg/dL. A modest agreement also existed for HbA1c and 2-h PG criteria for diagnosis of type 2 diabetes (κ coefficient = 0.427), with 81.8% of subjects classified as not having diabetes by both HbA1c and 2-h PG criteria, and 6.0% classified as having diabetes by both HbAc and 2-h PG criteria. Discordant classifications occurred for 5.3% of subjects with HbA1c ≥6.5% and 2-h PG <200 mg/dL and for 6.9% of subjects with HbA1c <6.5% and 2-h PG ≥200 mg/dL. Finally, moderate agreement existed for HbA1c and FPG and/or 2-h PG criteria for diagnosis of type 2 diabetes (κcoefficient = 0.446), with 80.1% of subjects classified as not having diabetes by both HbA1c and FPG and/or 2-h PG criteria, and 7.0% classified as having diabetes by both HbA1c and FPG and/or 2-h PG criteria. Discordant classifications occurred for 4.3% of subjects with HbA1c ≥6.5% and FPG <126 mg/dL and/or 2-h PG <200 mg/dL and for 8.7% of subjects with HbA1c <6.5% and FPG ≥126 mg/dL and/or 2-h PG ≥200 mg/dL (Figure 1).40 These results were in accord with those of two studies in adults from the US NHANES,36, 37 in a study in a cohort of older adults from the Rancho Bernardo Study,38 and in the Danish Inter99 study.39 In US adults from NHANES, moderate agreement was reported for HbA1c and FPG diagnoses (κcoefficient = 0.60), with 95.9% of the study participants classified as not having diabetes by both HbA1c and FPG and 1.8% classified as having diabetes by both HbA1c and FPG. Discordant classifications occurred for 0.5% of individuals with HbA1c ≥6.5% and FPG <126 mg/dL and for 1.8% of subjects with HbA1c <6.5% and FPG 126 mg/dL.36


Figure 1
Figure 1. Venn diagrams for diabetes.

Individuals meeting criteria for diabetes (HbA1c ≥6.5%; fasting plasma glucose
≥126 mg/dL; and 2-h postload plasma glucose ≥200 mg/dL).
Abbreviation: HbA1c, glycated hemoglobin.
Based on data from reference 40: Marini et al. Nutr Metab Cardiovasc Dis.
2012;22(7):561-566.



In the Rancho Bernardo Study comprising 2107 adults without known type 2 diabetes, a low agreement existed for HbA1c and FPG and/or 2-h PG criteria for diagnosis of type 2 diabetes (κ coefficient = 0.119), and 85% of participants with HbA1c ≥6.5% were classified as nondiabetic by FPG and/or 2-h PG criteria.38 The agreement for HbA1c and FPG criteria for diagnosis of type 2 diabetes was also low (κ coefficient = 0.061). The same pattern was observed considering diagnosis of type 2 diabetes based only on the 2-h PG criterion (κ coefficient = 0.112).

All together, these results suggest that individuals classified by HbA1c are different from those identified by the FPG or 2-h PG criteria. The discordance in the diagnosis of type 2 diabetes using diverse metabolic parameters is not completely unexpected because measurements of HbA1c, FPG, and 2-h PG, may reflect different features of glucose metabolism. Fasting hyperglycemia principally reflects hepatic insulin resistance and a dysfunction in the early phase of insulin secretion whereas postprandial hyperglycemia mainly reflects muscle insulin resistance and defects in late-phase insulin secretion.41 On the other hand, HbA1c may represent chronic exposure to both basal and postprandial hyperglycemia.41

Since there is only a modest concordance between the FPG and 2-h PG tests to diagnose type 2 diabetes, there is not likely to be perfect concordance between HbA1c and either glucose-based test for diagnosis of categories at increased risk for diabetes (also referred to as prediabetes), which includes those with IFG and IGT. Considering the expected increased utilization of HbA1c as a screening criterion, it is important to evaluate the concordance among the three tests to identify subjects at increased risk for type 2 diabetes. To this aim, we examined the concordance of HbA1c, FPG, and 2-h PG tests for the identification of prediabetes in a cohort of white Italians comprising 780 nondiabetic individuals.42 Low agreement was found for HbA1c and FPG criteria for identification of subjects with prediabetes (IFG) (κ coefficient = 0.332), with 56.3% of subjects determined to be without prediabetes according to both HbA1c and FPG criteria, and 15.8% classified as prediabetic by both HbA1c and FPG criteria. Discordant classifications occurred for 12.3% of subjects with HbA1c <5.7% and IFG and for 15.6% of subjects with HbA1c values of 5.7%-6.4%, and FPG <100 mg/dL. Low agreement existed for HbA1c and 2-h PG criteria for identification of subjects with prediabetes (IGT) (κ coefficient = 0.299), with 53.3% of subjects determined to be without prediabetes according to both HbA1c and 2-h PG criteria, and 16.4% classified as having prediabetes by both HbA1c and 2-h PG criteria. Discordant classifications occurred for 15.3% of subjects with HbA1c <5.7% and IGT and for 15.0% of subjects with HbA1c values of 5.7%-6.4%, and 2-h PG levels of <140 mg/dL. Finally, a modest agreement existed for HbA1c and FPG and/or 2-h PG criteria for diagnosis of prediabetes, with 46% of individuals classified as not having prediabetes by both HbA1c and FPG and/or 2-h PG criteria, and 10.4% classified as having diabetes by both HbA1c and FPG and/or 2-h PG criteria. Discordant classifications occurred for 9.5% of subjects who had an A1C 5.7%-6.4%, and FPG <100 mg/dL and/or 2-h PG <140 mg/dL (Figure 2, page 99).42


Figure 2
Figure 2. Venn diagrams for prediabetes.

Individuals meeting criteria for prediabetes (HbA1c=5.7% to <6.5%; fasting plasma glucose = 100 to 125 mg/dL; and 2-h postload plasma glucose = 140 to 199 mg/dL). Abbreviations: HbA1c, glycated hemoglobin; IFG, impaired fasting glucose;
IGT, impaired glucose tolerance.
Based on data from reference 42: Marini et al. Diabetes Care. 2012;35(5):
1144-1149.



Conclusions HbA1c levels reflect chronic glucose exposure, and are habitually utilized in monitoring glycemic control in order to guide therapy. HbA1c testing offers some practical advantages over assessments of FPG or 2-h PG, including lower day-to-day variability, less perturbation during periods of stress or illness, and requirement of a nonfasting sample. However, individuals identified as having type 2 diabetes or prediabetes according to HbA1c measurement may be distinct from those identified according to FPG and 2-h PG criteria; thus, if HbA1c screening is extensively implemented, it may to some extent change the present epidemiological setting of these dysglycemic conditions. The final goal would be to have a single universal diagnostic test for diabetes. Unfortunately, increasing evidence suggests that ethnic differences in HbA1c values may exist, and that some clinical situations, such as anemia and hemoglobinopathies, interfere with accurate HbA1c measurement. Additionally, given the higher costs, which are unaffordable in many countries, recommendation to use HbA1c as the only diagnostic test for diabetes on a global scale is not feasible at present, and the glucose assay will continue to have an important role in the diagnosis of diabetes and prediabetes. _


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Keywords: fasting plasma glucose; HbA1c; oral glucose tolerance test; prediabetes; type 2 diabetes