The relationship between HbA_1c and mean blood glucose

It took almost a further decade before clinical studies started to emerge suggesting that the increased proportions of HbA_1c in diabetes patients could be used as a reliable index of glycaemic control over the preceding weeks and months. It is indicative of the tools available at that time to assess glycaemic control that glycated haemoglobin was compared with 24-hour urinary glucose excretions,5 plasma “glucose brackets”,6 daily mean plasma glucose7 and the area under the curve of the glucose tolerance test.8 Following these studies there was rapid acceptance of glycated haemoglobin as a useful tool to objectively assess the prior glycaemic control of patients with type 1 and type 2 diabetes.

In some respects the general approval given to the use of the test at that time was ahead of evidence that it reliably reflected the mean glucose of patients with diabetes. However, the relationship was able to be more clearly defined by both the feasibility study of the Diabetes Control and Complications Trial (DCCT) and the subsequent full trial that followed.9 ,10 This was because the patients in the two treatments groups (intensively treated and conventionally treated) not only had HbA_1c recorded at quarterly intervals, but had a 7-point (pre- and postprandial) glucose profile collected and subsequently measured by a laboratory.11 As the 1441 patients participated in the trial for an average of 6.5 years, it was possible to make 26 056 comparisons between HbA_1c and a full 7-point profile.12 The linear relationship found (that mean plasma glucose (MPG, mmol/l) = 1.98 × HbA_1c (DCCT)−4.29, r = 0.82) has since been used as the most accurate guide to clinicians and other healthcare workers when discussing glycaemic control with their patients.12 Nevertheless, the scatter of patients around this regression line meant that an individual with a MPG of, say, 10 mmol/l, could have an HbA_1c anywhere between about 6% and 11%, and this has obvious implications when using solely HbA_1c to set glycaemic targets. More recently, the single linear relationship between MPG and HbA_1c has been called into question in that it appeared different between the two treatment groups of the DCCT such that the MPG was 1.2 mmol/l lower at 7% HbA_1c in intensively treated patients than in conventionally treated patients, with the difference becoming 4.6 mmol/l at 11% HbA_1c.13 This inferred that the relationship between MPG and HbA_1c may not constant but could differ depending on the glycaemic control of the population being studied.

Between 2006 and 2007, patients have been recruited to the “mean blood glucose study”, which aims to establish the relationship between HbA_1c and blood glucose as definitively as possible.14 At the European Association for the Study of Diabetes conference in September 2007, preliminary results of the study in 427 patients were presented. The study used continuous glucose monitoring and traditional pre- and postmeal glucose meter testing to establish the mean glucose of the patients, and used a National Glycohemoglobin Standardization Program (NGSP)-certified method to measure HbA_1c. The relationship between MPG and HbA_1c was found to be closer than in the DCCT, with a correlation coefficient of 0.91, and more in keeping with the findings of a smaller recent study (r = 0.90) that also used continuous glucose monitoring.15 However, it remains to be seen from any final publication how representative these mean blood glucose study patients are of the diabetes population as a whole, since there were many exclusion criteria to the study that could have removed the very people who might account for much of the scatter around the regression line.

Time course of HbA_1c formation

Glycation of haemoglobin occurs over the entire 120-day lifespan of the red cell,16 but within this 120 days recent glycaemia has the largest influence on the HbA_1c value.17 Indeed, theoretical models and clinical studies suggest that a patient in stable control will have 50% of their HbA_1c formed in the month prior to sampling, 25% in the month before that, and the remaining 25% in months 2–4.18 This explains why, traditionally, HbA_1c has been thought to represent average glycaemia over roughly the last 6–8 weeks.19

Effect of glucose variability on HbA_1c

Until recently there has been very little evidence to establish whether or not two patients with the same mean blood glucose but very different glucose variability would have similar HbA_1c values. However, two recent studies—one using DCCT data—have shown that glucose instability seems to have little influence on the HbA_1c result, and rather that it is the mean glucose that appears to be the main determinant, not how that mean is arrived at.20 ,21

Microvascular complications

The microvascular (small vessel) complications of diabetes comprise retinopathy, nephropathy and neuropathy. Patients with diabetes who develop these conditions constitute a large proportion of all subjects who develop blindness, renal failure and/or require limb amputation. The DCCT found that when 1441 patients with type 1 diabetes were randomised to “intensive” rather than “conventional” treatment, their median HbA_1c was 7.3% compared with 9.1% throughout the 6.5 years average follow-up period. The subsequent risk of developing retinopathy in the intensively treated group was reduced by 76%, the risk of developing proteinuria was reduced by 54%, and the risk of clinical neuropathy was reduced by 60%.10 Looked at from the perspective of HbA_1c, the risk of microvascular complications in the two patient groups rose exponentially as the HbA_1c value increased,24 with no threshold—short of normal glycaemia—below which patients with type 1 diabetes did not develop microvascular complications at all.25

The publication of the UKPDS in 1998 confirmed that a relationship between HbA_1c and microvascular complication risk existed in 3867 patients with type 2 diabetes.22 The difference in HbA_1c between the intensive and conventional treatment groups was not as large as in the DCCT (HbA_1c 7.0% versus 7.9% over 10 years), but there was still a 25% reduction in microvascular risk. A subsequent analysis of the data has shown that when the two treatment groups are combined, then a similar exponential relationship between rising HbA_1c and rising microvascular risk exists as in the DCCT.26

After the end of the DCCT, 96% of the patients in the original study agreed to continue to be followed up in a new study known as the Epidemiology of Diabetes Interventions and Complications study.27 They were no longer in two separate treatment groups. In fact, following the outcomes of the DCCT, it was recommended that all patients follow an intensive treatment regime. It was therefore interesting that, out of a clinical trial scenario, the HbA_1c of the previously intensively treated patients rose to an average of approximately 8%, while that of the conventionally treated group tightened up to a similar value.27 Long-term follow-up of these patients has shown that the benefits of improved glycaemic control during the DCCT on the risk of microvascular complications are maintained in the long term despite the convergence of glycaemia at the end of the original DCCT trial.28 ^– 30 This observation—that glycaemia from several years previously influences subsequent long-term complication risk—has since been dubbed “metabolic memory”31 and has reinforced the importance of good glycaemic control as soon as possible after the diagnosis of diabetes in order to avoid subsequent problems.

Macrovascular disease

Large vessel (macrovascular) disease remains the major cause of morbidity and mortality in patients with diabetes, with those having type 1 diabetes being at as high a risk as those with type 2.32 ^– 34

In type 1 diabetes, the DCCT found an excess of macrovascular events in the conventional compared with the intensive group (40 versus 23), although this just failed to reach statistical significance (p = 0.08).35 The small number of events were undoubtedly related to the young age (median 27 years at entry) of the study population. Subsequently, the Epidemiology of Diabetes Interventions and Complications study has shown that during a mean 17 years of follow up of these patients, 46 cardiovascular disease events occurred in 31 patients who had received intensive treatment in the DCCT, as compared with 98 events in the 52 patients who had received conventional treatment, meaning that intensive treatment reduced the risk of any cardiovascular event by 42% (p = 0.02).36 Detailed analysis showed it was the mean HbA_1c value during the DCCT that explained a large part of this beneficial effect on cardiovascular risk. Again, like the development of microvascular complications, this showed how vital it is to have good glycaemic control early after a diagnosis of (at least) type 1 diabetes. Also, even though HbA_1c was unable to predict macrovascular complications during the original DCCT study period, it has recently been shown that mean blood glucose did predict macrovascular complications, and this again reasserts the need for early tight glucose management.37

In the UKPDS, the event rate among the patients with type 2 diabetes was higher than in the DCCT, but the HbA_1c separation between the two groups was less marked. Nevertheless, there was a suggestion of more myocardial infarctions among conventionally treated patients (p = 0.052).22 In a subsequent analysis, where the two treatment groups were combined, there was an overall relationship between rising HbA_1c and increasing risk of myocardial infarction.26

Even among individuals not known to have diabetes, a large epidemiological study has provided evidence that HbA_1c may also be a marker of cardiovascular risk within the general population.38

Glycaemic variability and complication risk

As mentioned above, the fact that HbA_1c can give an integrated measure of mean glucose can be an advantage by balancing out the glycaemic excursions of an individual. However, since there is evidence from basic clinical research that glucose variability could, itself, be an independent risk factor for diabetes complications,39 ,40 there have been concerns that this may not be reflected by HbA_1c measurement alone.41 As it transpires, analysis of the DCCT dataset using the 7-point glucose profiles during the study have not confirmed any risk conferred by glycaemic variability above that already predicted by the mean glucose.42 ,43 In the UKPDS, although blood glucose was not measured throughout the day it might have been expected for insulin-treated patients to have greater glucose oscillations than non-insulin-treated patients and so have a different rate of complications, but such a difference did not appear to exist. Together, it means that this potential limitation of HbA_1c may not be as relevant as first envisaged. Since it would now be extremely difficult to perform a prospective study looking at the effect of glucose variability on complication risk that could adequately take account of other confounders, such as antihypertensive, antiplatelet and lipid lowering drugs, it may be that this particular question will never be definitively answered.

In contrast to glucose variability, analysis of the Pittsburgh Epidemiology of Diabetes Complications Study has suggested that HbA_1c variability itself could contribute to an increased macrovascular risk amongst patients with type 1 diabetes, perhaps indicating that long-term fluctuations in glucose control influence complications more than short-term fluctuations.44

HbA_1c and hypoglycaemia

Hypoglycaemia is the main barrier that prevents patients with diabetes achieving “normal” glucose control.45 Unfortunately, it remains a very significant cause of physical morbidity46 and mortality47 in patients, and recurrent episodes are often associated with psychological, quality-of-life, driving and employment issues.48 Perhaps surprisingly, few studies have been able to establish a relationship between low HbA_1c values and an increased risk of hypoglycaemia, but the DCCT certainly demonstrated an exponentially increasing risk as HbA_1c values fell.10 ,49 ,50 A recent analysis of the same study has also shown that the mean glucose value and the degree of glucose variability in a patient each independently added to the predictive ability of HbA_1c.51 It should be noted, however, that there is much more to hypoglycaemia risk than just these glycaemic markers, with features such as a prior history of hypoglycaemia or prolonged duration of diabetes also exerting a large influence.

Abnormal haemoglobins

Normal adult HbA₀ glycates to form HbA_1c, but if an abnormal haemoglobin is present then a patient is likely to form other glycated products such as HbS_1c, HbC_1c and so on, either in addition to or instead of HbA_1c.66 Many glycated haemoglobin analysers can now at least identify the non-glycated portion of these abnormal fractions,67 ,68 but there can still be difficulty in trying to make sense of what a patient’s HbA_1c would be were it not for their haemoglobinopathy. This is even more difficult in patients who have homozygous haemoglobinopathies where there will be no HbA present at all. Some instruments that base their analysis on affinity chromatography can more easily identify glycation on any form of haemoglobin molecule as can immunoassay methods, but even then there is the suggestion that some abnormal haemoglobins glycate at a different rate to native HbA and so may give rise to misleading results.68 ,69

Small proportions of fetal haemoglobin persisting into adult life used to cause considerable problems with some glycated haemoglobin methods because they would co-migrate or co-elute with the glycated haemoglobin fraction leading to an overestimation of the HbA₁ or HbA_1c result.70 Modern instrumentation is now able to account for fetal haemoglobin and so this issue should now be only of historic interest.71

Anaemia

Iron-deficiency anaemia can lead to rises in HbA_1c of up to 2% that can then subsequently be reversed by iron treatment.72 ^– 76 The reason for this rise is not fully known, but since iron deficiency is such a common finding, especially in pre-menopausal women, it could influence the diabetes management of many patients. There is also some evidence in pre-menopausal women without overt anaemia that a low mean cell haemoglobin is also associated with higher HbA_1c values.77 Reassuringly, however, the overall relationship between MPG and HbA_1c in the population of pre-menopausal women participating in the DCCT was no different to that of men, suggesting that overt anaemia needs to be present to have marked effects on HbA_1c measurement.78

Haemolytic anaemia has the opposite effect to iron deficiency by reducing HbA_1c in affected individuals. This is simply a consequence of reduced red cell survival, meaning a reduction in the availability of haemoglobin for glycation.79 All causes of this form of anaemia (including immune haemolytic anaemia, haemoglobinopathies and chronic renal failure) are affected.

Effect of drugs and health conditions

Any drug which gives rise to haemolytic anaemia will have the same effect. High-dose aspirin, by forming acetylated haemoglobin, can lead to spurious rises in HbA_1c using some methods, but the effect is usually only apparent at doses (4 g/day) that are well in excess of that prescribed normally.80

Renal failure can have complex influences on HbA_1c formation and measurement. Patients can be iron deficient, exhibit haemolytic anaemia and have altered red cell survival. Compounding the problem is the fact that urea-derived isocyanate can lead to the formation of carbamylated haemoglobin, which can be indistinguishable from HbA_1c when using some glycated haemoglobin methods.80 However, for most patients the overall effect does not seem too substantial with most HbA_1c methods.81

Standardisation of HbA_1c

In the 1980s and 1990s an important issue for HbA_1c measurement was the lack of standardisation of the assay, and this meant that different analysers could have widely differing reference intervals and give varying results with patient samples.82

As the DCCT and the UKPDS used the same method of analysis in their studies, this was felt to be a useful method to harmonise results against. It also had the added attraction that it meant patients could have their HbA_1c results compared directly with those of the subjects who participated in the two trials.83 In order to develop this international harmonisation an extensive network of reference laboratories was established by the NGSP based in the USA. In the last decade this development has made great strides in making the HbA_1c results reported from different laboratories much more comparable.83 ^– 85

However, the HbA_1c results reported by this means were not the “true” HbA_1c values, but simply the best that 1980s technology could deliver when the DCCT study was conceived. In order to rectify this situation, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) developed a reference method for HbA_1c that first established reference material of purified HbA_1c and HbA₀, and then a highly specific reference method for their measurement.86 ^– 88 Given this increased specificity it is perhaps not surprising that the results using this technique are between 1.5% and 2% HbA_1c lower than the NGSP results that relate to the DCCT.89 As a result, the move over to using these numbers has been resisted in some quarters because of a fear of confusion between the two sets of values,90 with some evidence from previous changes in HbA_1c numbers that clinicians may either under treat or over treat patients because they are still using the older targets.91

This led to the suggestion that rather than moving to these IFCC values, there could be a wholesale change to expressing HbA_1c as a “mean plasma glucose equivalent”,92 or, as it has recently been coined, “estimated average glucose” (eAG). The main purported reason for making this change is that patients would be able to equate this result more closely to that obtained from their blood glucose meter readings, although this move inherently means there will not be a single global result, as the eAG value will be different depending on whether glucose is expressed as mmol/l or mg/dl. In order for eAG to be a suitable alternative to HbA_1c in the first place, there has to be certainty that HbA_1c can truly give an accurate reflection of mean blood glucose in most situations. The mean blood glucose study described above is being used for that purpose. Little has yet been published on this study, but the a priori criteria for acceptable agreement is that at least 90% of patients should have a mean glucose that is within 15% of that derived from the simple regression of MBG on HbA_1c for the study population (ie, within 15% of the eAG). However, even if this criterion is fulfilled it equates to 99% of the population being within ±24% of the estimated average glucose, meaning that in two individuals with an eAG of 10 mmol/l, one patient could have an actual mean glucose of 7.6 mmol/l and another 63% higher at 12.4 mmol/l. Thus, the final results of the study will need to be examined closely to be sure that eAG is not adopted to the detriment of these types of patients.

Since this proposal, the IFCC has taken steps to make sure there is little chance of confusion between its results and those of DCCT values by changing its units from percentages to being expressed as mmol HbA_1c/mol HbA₀.93 ,94 The relationship between percentage and mmol/mol is one of a 10-fold change meaning that, for example, 7% by the IFCC reference system equates to 70 mmol/mol. This would obviously be a large upheaval for users of the test although it is sweetened, perhaps, by the fact that HbA_1c results could no longer be confused by patients as being their glucose result when expressed in SI units. Unfortunately, for those countries who express glucose as mg/dl it introduces this problem where it did not exist previously.

So where from here? Following a meeting on 4 May 2007, a joint consensus statement was issued by the European Association for the Study of Diabetes, the American Diabetes Association, the IFCC and the International Diabetes Federation. It recommended that HbA_1c results be reported worldwide in IFCC units (mmol/mol) and NGSP (ie, DCCT) units (percentage) and, if the ongoing mean blood glucose study fulfils its a priori criterion then the eAG will also be reported as an interpretation of the HbA_1c result.95

This author is bemused at how a single test can end up having to be reported in three different ways (four if glucose units are included) on the same report. It therefore seems inevitable that there will be a re-evaluation of this consensus at some stage, with the fear that there may be less rather than more global harmonisation of results in the future if individual groupings of countries choose to take different paths.