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Consensus Statement
Glycohaemoglobin: a crucial measurement in modern diabetes management Progress towards standardisation and improved precision of measurement*
Peter G Colman, G Ian Goodall, Peter Garcia-Webb, Paul F Williams and
Marjorie E Dunlop
MJA 1997; 167: 96-98
| Abstract |
Synopsis
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| Introduction | The landmark Diabetes Control and Complications Trial (DCCT)1 has focused increased attention on the importance of glycaemic control in preventing or retarding the progression of complications in patients with diabetes.2 Regular measurement of glycohaemoglobin is now recognised as an essential adjunct to self-measurement of blood glucose in achieving the best possible glycaemic control. However, clinicians using glycohaemoglobin assays should be aware of several potential problems which can confound the interpretation of the glycohaemoglobin result. | ||
| What is glycohaemoglobin and why should we measure it? |
Glycohaemoglobin (GHb) is formed by a non-enzymatic interaction
between glucose and the amino groups of the valine and lysine residues
in haemoglobin. Formation of glycohaemoglobin is irreversible and
the level in the red blood cell depends on the blood glucose
concentration. Thus, measuring glycohaemoglobin provides a
measurement of glycaemic control over time, and its use has been
proven to evoke changes in diabetes treatment, resulting in improved
metabolic control.3 First introduced in the
1970s, it is now accepted as a unique and important index of metabolic
control and was a major outcome measure in the DCCT.1
In the DCCT, 1441 patients with insulin-dependent diabetes were randomly allocated to intensive treatment and monitoring (usually with four insulin injections a day or pump treatment) with the aim of achieving normoglycaemia or to conventional treatment (usually with one or two injections a day). The effectiveness of intensive therapy was reflected in clear differences in mean blood glucose and glycohaemoglobin levels between the two groups. The intensive treatment group achieved a mean daily blood glucose level of 8.6 mmol/L and a median HbA1c value of 7.2% compared with the conventional treatment group, which achieved a mean blood glucose level of 12.8 mmol/L and a median HbA1c of 8.9%. These differences in glycaemic control were maintained over a mean period of 6.5 years and were associated with a 35%-76% reduction in retinopathy, nephropathy and neuropathy. Using the knowledge gained in the DCCT, doctors caring for patients with diabetes can now establish targets for glycaemic control that are based on observed outcomes, and which, if met, should minimise the development of complications. Inevitably, because glycohaemoglobin measurements reflect an integrated view of glycaemic control over time, the patients and their carers will place increasing reliance on the glycohaemoglobin result. So it is timely to evaluate the types of assays available, the moves toward standardisation of the reporting units and the precision and reproducibility of current assays. In the DCCT all glycohaemoglobin measurements were performed using the same closely standardised method. Unfortunately, in Australia there are currently four principal glycohaemoglobin assay techniques and about 20 different specific methods, most of which are not standardised between laboratories. |
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| Types of assays available |
The four principal techniques used to measure glycohaemoglobin are
ion-exchange chromatography, electrophoresis, affinity
chromatography and immunoassay. The techniques measure slightly
different glycated products and use at least three different units
for reporting the results (%HbA1c, %HbA1 and % total
GHb). They can produce different values for the same patient
specimen.
This was demonstrated in a recent study in which four whole blood samples with HbA1c levels of 5.1% (representing non-diabetes), 6.7% (representing excellent glycaemic control), 8.5% (representing moderate glycaemic control) and 11.4% (representing poor glycaemic control) were distributed to 29 laboratories in Victoria for glycohaemoglobin determinations.4 The range of values obtained for the non-diabetic (4.1%-6.8%), good control (5.1%-9.3%), moderate control (6.7%- 11.9%) and poor control (10.1%-17.3%) specimens demonstrated extensive overlap between measurements of samples from patients with markedly different degrees of glycaemic control (Box 1). At present it is impossible to compare the results from two different laboratories; this can be confusing not only for patients but also for their carers. Laboratory- specific reference ranges are a means by which results from different laboratories can be compared, but the data used to derive such ranges are arbitrary and the categories into which different glycohaemoglobin levels are divided may be misleading. |
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| Importance of reproducible measurement |
A major use of the glycohaemoglobin assay is to assess changes in
metabolic control that follow an alteration in treatment. The
ability of any assay to reliably detect a change depends on its
reproducibility (the ability of the assay and laboratory to get the
same answer for the same sample each time). Reproducibility is
normally expressed as the coefficient of variation (CV) of an assay.
The CV is obtained by measuring the same sample at least 20 times in
different assay runs and calculating the mean and standard deviation
(SD) of the measurements; the CV is calculated by dividing the SD by the
mean and expressing the result as a percentage. An assay with a high CV
suffers from poor reproducibility and cannot demonstrate whether
glycohaemoglobin levels have changed in different samples.
Laboratories normally accept an assay for reporting purposes if the result for quality control samples falls within three SDs of the mean (3SD range). The imprecision of measurement of patient samples will be similar to that of the quality control samples. For example, if the result of an HbA1c assay with good precision (3% CV) was 7%, the 3SD range would be 6.37%-7.63%; for a result of 9%, the 3SD range would be 8.19%-9.81%. These two results can clearly be separated. In contrast, the same results of an assay with poor precision (6% CV) would have 3SD ranges of 5.74%-8.26% (for the 7% level) and 7.38%-10.62% (for the 9% level), and could not be differentiated. |
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| How reliable are assays in Australia? |
The Royal College of Pathologists of Australasia/Australasian
Association of Clinical Biochemists Chemical Pathology Quality
Assurance Programme provides external quality control samples for
Australian laboratories that report glycohaemoglobin
levels.5 The program runs on a
six-monthly cycle, in which participating laboratories analyse two
random samples per month, drawn from lyophilised whole blood samples
representing six levels of glycohaemoglobin. The use of lyophilised
samples can lead to minor variations in assay values for some methods.
However, a recent study has excluded this as a complicating
factor.6
When measuring control samples with the value of 7.2% HbA1c (the mean outcome of intensive treatment in the DCCT), Australian laboratories reported HbA1c assay results between 6% and 9% HbA1c, while the range of values reported for all units (percentage of HbA1c, HbA1 and total GHb) was between 6% and 12.6%. When measuring control samples with the value of 8.9% HbA1c (the mean outcome level for conventional treatment in the DCCT trial), laboratories reported HbA1c values between 7.4% and 11%, while the range of values for all glycohaemoglobin units was 7.4% to 16.4%. The overlap between values obtained for these samples epitomises the problems currently facing clinicians in interpreting glycohaemoglobin levels and changes in levels reported by different laboratories. The interlaboratory CV obtained varied between 1.6% and 8.9% for the most common assays. To critically evaluate changes in HbA1c, the precision of individual laboratory assays for glycohaemoglobin must be known. For example, the difference in mean HbA1c value between the intensive and the conventional treatment groups in the DCCT was only 1.7%, and any assay used should at least be able to detect a difference of this order. With most laboratories using the 3SD range to accept or reject assay runs, glycohaemoglobin assays with CVs close to 3% are necessary to differentiate the two DCCT group means (Box 2). At 3% CV, the 3SD range of values for a patient with a true HbA1c level of 8.05 %HbA1c would be 7.33 to 8.77 %HbA1c. This range is less than ideal, but, realistically, only high pressure liquid chromatography assays currently achieve such precision. We recommend that the CV of the assay currently being used by the reporting laboratory be made available to carers who use glycohaemoglobin measurements. This will allow them to determine if the assay has the ability to differentiate between reported levels. Reference laboratories in the International Federation of Clinical Chemistry (IFCC)/American Association of Clinical Chemistry (AACC) International Standardization Programme must be able to achieve a CV below 3% at HbA1c levels of 6% and 9%.7 Manufacturers' assays should be able to achieve a CV below 5%. Currently, some GHb assays are unable to achieve these limits. |
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| Progress towards standardisation | Standardisation is crucial to allow comparison of results obtained in different laboratories. A working party of the IFCC and AACC is coordinating an international effort by which all methods will be standardised to a designated method. This will be performed at the manufacturer level. Glycohaemoglobin analyser and kit manufacturers will have their assays standardised by reference laboratories established and monitored monthly by the IFCC/AACC working party. Thus, ultimately all laboratory methods will report their results in %HbA1c units which have been standardised against the DCCT method.8 Patients and carers will then be able to directly compare their level of glycaemic control against the enormous amount of data obtained by the DCCT trial on the onset and incidence of diabetes-related complications. | ||
| References |
* Consensus statement from the Australian Diabetes Society, the Royal College of Pathologists of Australasia and the Australasian Association of Clinical Biochemists
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Authors' details | |||
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Department of Diabetes and Endocrinology, Royal Melbourne
Hospital, Melbourne, VIC.
Peter G Colman, FRACP, MD, Director. Special Chemistry Unit, Austin and Repatriation Medical Centre,
Melbourne, VIC.
St John of God Pathology, Perth, WA.
Royal Prince Alfred Hospital, Sydney, NSW.
University of Melbourne Department of Medicine, Melbourne, VIC.
Reprints: Dr P G Colman, Department of Diabetes and
Endocrinology, Royal Melbourne Hospital, PO Box 3050, Parkville,
VIC 3050. ©MJA 1998
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| 1: Results of glycohaemoglobin assays of four samples in 29 Victorian laboratories4
The four samples were from patients with differing degrees of diabetes control. The closed circles represent individual laboratory results for each sample and the open circles represent the notional target value. The notional target value was set by the Biorad Diamat (Biorad Laboratories, Hercules, California) in a laboratory where the assay was referenced against the DCCT method.2 Six methods of measuring glycohaemoglobin were used by the laboratories:
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