Problems in the assessment of glycaemic control in diabetes mellitus

REVIEW

Problems in the Assessment ofGlycaemic Control in Diabetes MellitusE.S. Kilpatrick*

Department of Chemical Pathology, Withington Hospital,Manchester, UK

The measurement of glycated haemoglobin and serum fructosamine to assess the recentglycaemic control of diabetic patients has become well established. Likewise, the monitoringof blood glucose using glucose test strips and meters has become popular in both thecommunity and in the hospital inpatient environment. However, despite improvements inthe methods of analysis, clinically inaccurate assessments of glycaemia can still occur.Specific problems such as the lack of standardization in assays are in the process of beingresolved, but inherent difficulties associated with these measures remain. Clinicians shouldbe aware that these tests still need to be interpreted in conjunction with clinical prudence. 1997 by John Wiley & Sons, Ltd.

Diabet. Med. 14: 819–831 (1997)

No of Figures: 1. No of Tables: 3. No of Refs: 156

KEY WORDS Glycaemic control Glycated haemoglobin Fructosamine blood glucose

Received 14 January 1997; accepted 28 April 1997

Introduction Glycated Haemoglobin

The last 20 years have brought about a major advance Clinical Utilityin the assessment of glycaemic control in diabeticpatients. Two developments, in particular, have been Following the discovery that diabetic patients exhibited

an increase in some of the minor fractions of haemo-instrumental in this. The first has been the ability, sincethe 1970s, of patients to assess their own glucose control globin,3,4 it was established that this ‘diabetic haemo-

globin’ was structurally identical to the component ofthrough the self-monitoring of blood glucose (SMBG)using glucose test strips and portable meters. The second haemoglobin known as HbA1c.5 Haemoglobin A1c had

previously been described in non-diabetic adults,6 butis the subsequent development of glycated haemoglobinand fructosamine assays which have allowed clinicians in comparison to diabetic patients it was present in

much smaller proportions. Nearly a decade passed beforean independent means of judging a patient’s prior glucosecontrol. Although other indicators of glycaemia (such as a flurry of clinical studies showed that the increased

proportions of HbA1c in diabetic patients could be usedglycated albumin1 and 1,5-anhydroglucitol2) have beenproposed, they are not routinely used in clinical practice. as a reliable index of glycaemic control over the preceding

6–8 weeks. The studies demonstrated correlation ofThis ability to accurately assess present and priorglucose control has undoubtedly benefited the treatment glycated haemoglobin with other known indicators

of diabetic control such as 24-hour urinary glucoseof patients with diabetes. Clinicians can now set goalsof glycaemic control for their patients by using glycated excretion,7 plasma ‘glucose brackets’,8 daily mean plasma

glucose9 and the area under the curve of the glucosehaemoglobin or fructosamine values while patients canempower themselves to meet these aims through the tolerance test.10 Following these studies, and despite

considerable methodological difficulties in measurement,self-monitoring of blood glucose. However, errors in anyof these measurements may either lead to the setting of acceptance of the use of glycated haemoglobin by

diabetologists and other healthcare workers was rapidunattainable goals or conversely the acceptance ofspuriously good glucose control. because for the first time they had an apparently simple

tool which could give a completely objective assessmentAs these measurements become further established itis easy to assume that any faults which may have existed of a patient’s glucose control.11 Thus treatment regimes

could now be changed without relying solely on amust now have been removed. This article reviews theclinical utility, the analytical methods and the problems patient’s description of symptoms or home urine/blood

test results.associated with using glycated haemoglobin, serumfructosamine, and glucose test strips. As well as its use in monitoring diabetic patients, it

was hoped that measurement of glycated haemoglobincould also be used as an alternative to the laborious* Correspondence to: Dr E.S. Kilpatrick, Department of Chemical

Pathology, Withington Hospital, Manchester M20 2LR, UK and inconvenient glucose tolerance test (GTT) in diag-

819CCC 0742–3071/97/100819–13$17.50 1997 by John Wiley & Sons, Ltd. DIABETIC MEDICINE, 1997; 14: 819–831

REVIEWnosis. Even following the widespread recognition of ive haemoglobins by charge-dependent techniques. In

contrast, glycation at sites other than the b-chain aminoWHO criteria for the GTT, and the undertaking ofnumerous studies, there continues to be some debate as terminus results in a compound with a charge not

dissimilar to non-glycated haemoglobin and so is indis-to the usefulness of glycated haemoglobin in diagnosisand screening. A recent review of 25 studies confirmed tinguishable by these methods. However, this portion,

which accounts for about half of all haemoglobinthat in a subject with a mildly elevated glycatedhaemoglobin level the test was still not specific or glycation, can be detected if glycation-specific ‘total

glycated haemoglobin’ methods such as affinity chroma-sensitive enough to distinguish reliably between nor-mality, impaired glucose tolerance or diabetes.12 tography are used (see below).

The charge-separated haemoglobins of normal adultFollowing the first clinical studies it was realized thatglycated haemoglobin measurement could prove to be HbA0 are jointly known as haemoglobin A1 (HbA1).

Improved techniques have allowed further separation ofuseful in answering one of the fundamental questionsin diabetes, namely, whether long-term microvascular HbA1 into its constituent parts, HbA1a1, HbA1a2, HbA1b,

and HbA1c. Glucose is the carbohydrate in the majorcomplications of the disease related to the degree ofcontrol of hyperglycaemia.13 Although early studies had fraction, HbA1c, while other carbohydrates, some of

which have still to be established with certainty, constitutesuggested such a link,14,15 the technical advances ofglycated haemoglobin, capillary blood glucose measure- the other fractions (Table 1).23.

Glycation of haemoglobin occurs continuouslyment, and intensified insulin regimes now made itpossible to conduct more meaningful trials.16 Indeed, throughout the 120-day lifetime of the red cell,24 so the

eldest cells will be most glycated and the youngestonce available, glycated haemoglobin measurementbecame the cornerstone of treatment evaluation in all least.25 However, all ages of cells will have been exposed

to recent levels of glycaemia while only the eldest cellssuch studies.17 In particular, the Diabetes Control andComplications Trial (DCCT) showed conclusively that will also have been exposed to glucose levels from 4

months previously. Therefore, the more recent the periodglycaemic control as assessed by glycated haemoglobincan predict the risk of developing microvascular diabetic of glycaemia, the larger its influence will be on the

glycated haemoglobin value. Consequently, it has beencomplications in Type I patients.18 As a consequence,the usefulness of the assay was vindicated and its suggested that half of a HbA1c value is attributable to

changes in glycaemia over the preceding month, a furtherincreased use recommended.16,17,19,20 Moreover, it wassuggested that the cost of using the test in routine clinical quarter is due to the month prior to that, with the

remaining quarter a reflection of months 3 and 4.26practice was likely to be offset by much larger savingsdue to the decreased need for procedures such aslaser photocoagulation therapy, renal dialysis, and renal Analytical Methodstransplantation.16 The continued use of glycated haemo-globin measurement in diabetes assessment would thus Methods in routine use for the measurement of glycated

haemoglobin separate the molecule on the basis of eitherseem assured.its charge, or its structure or its antigenic properties.Table 2 gives the methods and instruments that are inGlycated Haemoglobin: Structure androutine use as described by the Wales External QualityFormationAssessment Scheme (WEQAS).

The most popular means of measurement rely onHuman haemoglobin demonstrates marked heterogen-eity, mainly as a consequence of post-translational the increased negative charge found in the glycated

haemoglobin molecule to distinguish it from its non-changes due to the non-enzymatic binding of variouscarbohydrates in a process known as glycation. Glycation glycated form. These assays include electrophoresis and

ion-exchange chromatography.27 The latter method isoccurs via a carbohydrate such as glucose reacting inits free aldehyde form with a haemoglobin molecule to becoming increasingly popular with the development of

rapid (as short as 2 min between samples) dedicated highform the Schiff-base compound, aldimine.21 The formedaldimine can then meet one of two further fates: it caneither dissociate back to its component carbohydrate Table 1. Carbohydrates involved in the formation of charge-and haemoglobin or, in a process that is 60-fold less separated haemoglobins with their approximate abundance in

non-diabetic individualsrapid, it can undergo an intermolecular transformationknown as the Amadori rearrangement to form a stable

Haemoglobin Modification Abundance (%)glycated ketoamine product.Haemoglobin glycation occurs at several sites, namely,

Ao 95the amino terminal of both its a and b-chains, as wellHbA1a1 fructose-1,6-diphosphate 0.2

as certain e-amino groups.22 However, it is only the HbA1a2 glucose-6-phosphate 0.2modification at the N-terminal valine amino acid of the HbA1b carbohydrate (?) 0.5HbA1 5HbA1c glucose 4b-chain which imparts enough negative charge to thehaemoglobin molecule to allow separation of the respect-

820 E.S. KILPATRICK

Diabet. Med. 14: 819–831 (1997) 1997 by John Wiley & Sons, Ltd.

REVIEWTable 2. Glycated haemoglobin methods in routine laboratory fully in diabetic clinics.34 Its assay is based on latexuse as measured by involvement in the Welsh External immuno-agglutination inhibition methodology and usesQuality Assessment Scheme

a disposable cartridge to measure each sample individu-ally. In a situation similar to the affinity chromatographyHbA1c HbA1assays, all these immunoassay methods are calibratedwith material to which HbA1c values have been assignedIon exchange HPLC Ion exchange HPLC

Biomen HA 8121 Biomen HA 8121 using ion-exchange HPLC.Biorad Variant Corning GlycomatBiorad Diamat

Electrophoresis Factors Influencing Glycated HaemoglobinCorning Glycomat 745/765Helena RepShimadzu Measurement

Affinity chromatography Lack of StandardizationAbbott IMx

With such a wide range of instruments and methodsGamidor Primusavailable for glycated haemoglobin measurement it is

Affinity electrophoresis perhaps not surprising that instruments which measureBeckman Diatrac

different species (such as either HbA1, HbA1c or totalImmunochemical glycated haemoglobin) produce results which are not

Bayer DC 2000 directly comparable. However, due to a lack of stan-Boehringer Mannheim Tinaquant

dardization, even methods which purport to measureDako Novaclonethe same analyte can have widely differing referenceRoche Unimateintervals and give varying results with patient samples.Indeed, returns to UK external quality control schemeshave shown poor between-laboratory agreement forglycated haemoglobin, with an overall coefficient ofperformance liquid chromatography (HPLC) instruments

which are much less labour intensive than electro- variation of 20 %.35 As such, this lack of standardizationis regarded as one of the major drawbacks that limitsphoresis. These new analysers have also made feasible

the measurement of glycated haemoglobin ‘on-site’ at the clinical utility of glycated haemoglobin measure-ments,36 and both British Diabetic Association37 and thediabetic outpatient clinics,28 allowing ‘near patient’ test-

ing. National Diabetes Data Group38 see solving the problemas a major priority.Boronate affinity chromatography separates glycated

haemoglobin on the basis of its structure rather than A number of different approaches have been used toaddress the issue. European guidelines (established beforecharge. In this assay, separation occurs by the carbo-

hydrate moieties present on glycated haemoglobin bind- the DCCT report) have suggested that glycaemic controlbe classified according to how many standard deviationsing by condensation to the affinity reagent, di-hydrox-

yboronate.29 This method is specific for all glycated (SDs) a patient’s HbA1 or HbA1c lies from the non-diabetic mean value for the particular assay.39 Withinhaemoglobins irrespective of molecular charge or the

site of glycation on the haemoglobin molecule. In 3SD of the non-diabetic mean is defined as good control,between 3 and 5SD is borderline, and outwith 5SD isaddition, it is also able to detect the glycated portion of

haemoglobin in patients with haemoglobin variants such poor. However, these guidelines have a number ofdeficiencies, not least the fact that HbA1 assays classifyas HbS, HbC or HbF. Thus, the term ‘total glycated

haemoglobin’ has been used in describing this type of patients markedly different from those of HbA1c.40

Other groups have proposed alternatives such asassay. Nevertheless, despite some reservations,30 theseglycated haemoglobin values are usually expressed as comparing local laboratory values with those obtained

when the same samples are measured by the DCCT‘HbA1c equivalents’ by comparison with those obtainedusing an ion-exchange HPLC instrument. Some affinity central HbA1c laboratory.41 This pragmatic approach of

using a ‘designated comparison method’ is being activelychromatography methods have now been automated31

and made faster by the development of affinity HPLC sys- pursued by Goldstein and colleagues in the UnitedStates.42 The use of lyophilized calibrators to calibratetems.

Immunoassays have also been developed for the local instruments to produce similar values has also beenused with a degree of success in the Netherlands.43,44measurement of HbA1c. The first, the Dako Novoclone,

was based on an enzyme immunoassay performed on However, it has not been without problems. For example,lyophilized specimens were found to be incompatiblemicrotitre plates employing a monoclonal antibody

raised against HbA1c.32 More recently, high throughput with total glycated haemoglobin assays and delay in thetransport of samples between laboratories producedimmunoturbimetric methods have been developed which

can be adapted for use on a number of clinical chemistry variations in results. In addition, all these proposedschemes, with the exception of the European guidelines,analysers.33 However, such methods still require labora-

tory analysis, but a portable instrument, the DCA 2000 have the disadvantage of being time-consuming andexpensive to maintain.analyser from Bayer Diagnostics, has been used success-

821ASSESSMENT OF GLYCAEMIC CONTROL

1997 by John Wiley & Sons, Ltd. Diabet. Med. 14: 819–831 (1997)

REVIEWIt seems likely that all these suggestions will be globinopathies will fail to detect any glycated haemo-

globin and will necessitate measurements of derivativessuperseded by work being performed by the WorkingGroup on Standardization of HbA1c on behalf of the such as HbS1c or HbC1c. In heterozygous haemo-

globinopathies (e.g. HbAS, HbAC) or haemoglobin syn-International Federation of Clinical Chemistry (IFCC).45

Their approach is fundamental rather than pragmatic. thesis variants (e.g. b-thalassaemia), the same specificassays are likely to underestimate the degree of haemo-Thus, instead of comparing or calibrating equipment to

an arbitarily chosen instrument, for the first time a globin glycation by an amount dependent on the ratioof normal:abnormal haemoglobin. Examples of thesescientifically based reference system is to be established.

The reference material is to consist of highly purified specific methods include the electrophoretic, HPLC, andthe previously mentioned immunoassay methods. InHbA1c and HbA0 while the reference method consists of

measuring glycated and non-glycated peptides by a some of these systems the problem is easily identifiedbecause the abnormal haemoglobins separate to formhighly specific HPLC-mass spectrometry technique. This

system will then be used to produce calibrators for additional peaks, but in other HPLC and immunoassaytechniques these abnormal haemoglobins may be indis-routine methods. Values obtained by this system are

likely to differ from methods used in major clinical trials tinguishable from HbA0.48 The only assays able to givemeaningful measurements in these conditions are thosesuch as the DCCT, so ‘translation’ of these results will

be an important task for the IFCC group. based on affinity chromatography because of their abilityto measure glycation at any site of any haemoglobinThe full standardization of glycated haemoglobin

measurements as described is not expected until at least molecule. However, even measurement of this ‘totalglycated haemoglobin’ appears to be problematic sincethe end of the decade.45 In the meantime, proposals

such as the European guidelines are likely to continue it would seem that haemoglobins S and C glycate fasterthan native HbA0, thus leading to possible overestimationto be the only means of comparing results obtained from

different laboratories using differing methods. of the glycated haemoglobin value in the heterozygous(e.g. HbAC) state.49

HaemoglobinopathiesNormal adult haemoglobin is assembled from a-, b-, Fetal Haemoglobin

Fetal haemoglobin (HbF) has relevance to the measure-g-, and d-chains to form HbA0 (a2b2), fetal haemoglobinHbF (a2g2) and HbA2 (a2d2).24 As described above, ment of glycated haemoglobin beyond that of abnormal

haemoglobins. It is present physiologically as 75–100 %glycation of normal adult haemoglobin (HbA0) leads tothe formation of HbA1 and HbA1c. However, point of total haemoglobin in newborn children then declines

during childhood to less than 1 % in most adults.50mutations in the primary structure of the b-chain cangive rise to structural variants of haemoglobin, such as However, for reasons unknown, this decline can be

variable and incomplete, even in patients known not tothose found in HbS (b6 glu→val), HbC (b6 glu→lys), and HbE(b26 glu→lys). Also, impaired synthesis of b-chains leads have hereditary persistent HbF. The finding would be of

no consequence was it not for the fact that HbF, whetherto conditions such as b-thalassaemia and hereditarypersistent HbF. Patients with any of these haemo- glycated or not, co-migrates or co-elutes with the glycated

haemoglobin peak of many currently used electrophoreticglobinopathies (Table 3) are likely to form glycatedproducts such as HbS1c, HbC1c or HbE1c either in addition and HPLC methods.51,52 Thus, any detectable percentage

of HbF in a patient sample is likely to be included into or instead of HbA1c.46

The influence of abnormal haemoglobins on glycated the HbA1 or HbA1c result of these methods leading tofalsely elevated results.51,53,54 Moreover, compared tohaemoglobin measurement is variable depending on

the method of analysis used.47 In general, specific non-diabetic controls, increased concentrations of HbFhave been found in adult insulin-dependent and insulin-measurements of HbA1 or HbA1c in homozygous haemo-treated subjects thereby exacerbating the problem in thesepatients.55–57 Together, these findings have promptedTable 3. Factors potentially influencing glycated haemoglobin

measurements independent calls for the withdrawal of all glycatedhaemoglobin methods where fetal haemoglobin is a

Underestimation of Haemoglobinopathiesa, e.g. HbS potential interferent in measurement58,59 and, it is hoped,HbA1 or HbA1c Impaired haemoglobin synthesis e.g. have alerted clinicians and equipment manufacturers to

b-thalassaemiathe unacceptability of their continued use.Reduced red cell survival

Haemoglobin DerivativesOverestimation of HbF

Haemoglobin derivatives arise from post-translationalHbA1 or HbA1c Uraemiamodification of haemoglobin. Examples are thoseHigh-dose aspirin

Increasing age resulting from reactions with glucose (to form glycatedhaemoglobin), urea-derived isocyanate (to form carbamy-

Consult your local laboratory for details as errors are method and lated haemoglobin) and acetylsalicylic acid (to forminstrument dependent.

acetylated haemoglobin).60 As is the case for abnormalaSome haemoglobinopathies may lead to an overestimation when usingtotal glycated haemoglobin methods. haemoglobins, glycated haemoglobin methods are affec-

822 E.S. KILPATRICK


REVIEWted differently by the latter two derivatives. Both electro- to use an alternative method of glycaemic control

assessment such as the measurement of serum fructosami-phoretic and HPLC methods show increases in glycatedhaemoglobin values with increasing degrees of uraemia ne.75

as a consequence of rising concentrations of carbamylatedhaemoglobin.61–63 For approximately every 15 mmol l−1

Serum Fructosamineof urea, glycated haemoglobin measurements increaseby 1 %.63 In contrast, affinity chromatography and Clinical Utilityenzyme immunoassay methods seem free from inter-ference.63,64 Acetylated haemoglobin affects the same

By contrast with glycated haemoglobin, serum fructosam-methods as the carbamylated form, although in patientsine measurement was quickly estabished as a usefulon chronic low doses of aspirin (200–300 mg day−1), theindicator of a patient’s glycaemic control over theeffect would appear negligible.63 However, in rheumatoidprevious 1–3 weeks. Johnson, Metcalf and Baker frompatients receiving 4 g day−1, rises in glycated haemoglobinNew Zealand were the first investigators to propose theof 1.5 % have been found when using HPLC methods.65

use of a simple colorimetric assay for the assessmentAge-related changes ‘serum glycosylprotein’ concentrations in 1982.76 They

Glycated haemoglobin values in non-diabetic individ- based their assay on the ability of serum proteinuals appear to increase with subject age,66,67 while those ketoamine linkages to glucose being able to act asof serum fructosamine and fasting plasma glucose do reducing agents in alkaline solution. These proteinnot.67 Although the cause of this remains speculative, ketoamines were generically termed ‘fructosamines’, notthe finding nevertheless has a number of important because of the involvement of fructose but because theimplications. Firstly, it means that HbA1c reference resulting compound had structural similarities to thisintervals are likely to be influenced by the age of the sugar.77 Since all serum proteins can form ketoaminessubjects chosen. This in turn adds a further complication through glycation, fructosamine was found to give ato methods (such as the European guidelines) which similar indication of glycaemic control to that foundattempt to assess glycaemic control by comparing diabetic when measuring total glycated protein and glycatedHbA1c values with those from non-diabetic subjects. albumin.78–81 However, in contrast to assaying the latter,Secondly, if, compared to younger individuals, the fructosamine could be measured by spectrophotometerglycated haemoglobin values from elderly subjects are instruments found in most clinical biochemistry labora-closer to those of diabetic patients then it may also tories. Therefore, the assay initially gave the promise ofaccount for the finding that HbA1c is a better screening being a less expensive and quicker alternative to thetest for diabetes in middle aged rather than elderly majority of glycated haemoglobin analyses methods usedpeople.68 Lastly, since it would seem appropriate that at the time.82,83 Indeed, early cross-sectional studiesdiabetic patients should have their glycated haemoglobin seemed to show good correlations between fructosaminevalues compared to those of their non-diabetic chrono- and glycated haemogobin,82–86 but subsequent studieslogical peers, then age related HbA1c reference intervals have since shown that marked discrepancies can existmay be required to facilitate more accurate glycaemic between the two measures,87–92 not least because theycontrol targets and for the better auditing of a clinic per- measure glycaemia over different time-scales. Neverthe-formance.67

less, the shorter time period measured by fructosaminehas especially lent itself to use in pregnant diabeticLabile Haemoglobinpatients93 where frequent objective monitoring is essentialMany original electrophoretic and liquid chromatogra-for the health of both mother and fetus.94,95phy methods for measuring glycated haemoglobin

Fructosamine, like glycated haemoglobin, has alsoshowed marked fluctuations in results with acute changesbeen evaluated as a tool for the diagnosis of diabetes.in blood glucose.69,70 This was due to these methodsAfter initial optimism96,97 studies have since found it tobeing unable to distinguish between HbA1/HbA1c andlack the sensitivity and specificity required of a screeningthe intermediate aldimine or Schiff base.71 This lattertest.98–102 Moreover, as a substitute for the GTT, it seemscompound, also known as the labile haemoglobin fractionto be inferior to measuring glycated haemoglobin.101,102or pre-HbA1c, has for over a decade been removed

chemically72 and should now only be of historical interest.

Fructosamine MethodsDecreased Red Cell SurvivalProcesses which lessen mean red cell life to less than

120 days will reduce the availability of haemoglobin for The fructosamine assay relies on the ability of serumAmadori compound to directly reduce nitroblue tetrazol-glycation. As a consequence, lower glycated haemoglobin

values have been recorded in patients with chronic ium (NBT) to the tetrazinolyl radical NBT+, whichdisassociates to yield a highly coloured formazan dye inrenal failure73, immune haemolytic anaemia,74 and

homozygous haemoglobinopathies.75 This phenomenon an alkaline environment.103 The reaction rate is followedby measuring the increase in dye absorbance at 530 nmis independent of the method of analysis used and

cannot be easily corrected, so often the only recourse is wavelength. While NBT is reduced, the oxidation



REVIEWdegeneration product of the Amadori compound would Clinical evidence would also support the fact that,

even with improvements in the assay, pathologicalappear to be d-glucosone.104

In 1991 the commercial assays from Boehringer changes in serum proteins can affect fructosamine.112

For example, patients with cirrhosis of the liver, nephroticMannheim and Roche underwent modifications byincluding a detergent and uricase in the reagent, and by syndrome113,114 and hyperthyroidism (associated with

increased protein turn-over)115 all demonstrate reducedincreasing the concentrations of both the carbonatebuffer and the dye.105 This helped reduce the effect of values. Even normoalbuminaemic Type 1 diabetic

patients have shown diurnal variation in fructosaminelipaemia and hyperuricaemia in samples as well asdiminishing protein matrix effects.106 At the same time, levels closely related with changes in their serum protein

concentration.116 However, there has been consensusthe detection wavelength of absorption changed from530 to 550 nm and glycated polylysine replaced deoxy- agreement that in patients with a serum albumin concen-

tration greater than 30 g l−1, the effect will be negligible.112morpholinofructose as the assay calibrant.107 Toaccompany these wholesale changes, the units for It is generally assumed that albumin makes the largest

contribution to total glycated protein concentration, butfructosamine measurement changed from mmol l−1 tommol l−1 with the results for the two assays not being it has also been suggested that certain proteins such as

IgA may have a greater influence on fructosamine values,directly comparable.108

presumably because they glycate at a different rate. Thisdisproportionate contribution of IgA, either in healthyFactors Influencing Serum Fructosaminesubjects117 or in patients with paraproteinaemias,118 mayMeasurement thus partly explain why the influence of serum proteinsin fructosamine measurement remains controversial.Lack of Standardization

Calibration of the fructosamine assay has proved to Interfering Substancesbe a major problem because of the lack of a trustworthy The original fructosamine assay was subject to inter-standard or calibrator. The obvious candidate, glycated ference from reducing substances other than ketoamineserum albumin, has proved to be extremely difficult to linkages, especially uric acid and ascorbic acid. Commonmanufacture to a known and reproducible amount.109 photometric interferents such as bilirubin, haemolysis,Even if feasible, storage in a matrix suitably similar to and lipaemia also interfered. Bilirubin and lipaemiahuman serum would pose further difficulties. As an caused increased fructosamine values while haemolysedalternative, the original fructosamine assay used the samples produced falsely low ones.77 The introductionless desirable deoxymorpholinofructose (DMF). Although of new fructosamine kits not only changed the calibrators,similarities between the reaction of NBT with DMF and but also addressed hyperuricaemic samples by includingwith human serum existed, the reaction kinetics and a uricase, and lipaemic samples by including surfactantabsorption spectra were markedly different. The situation detergents. Increasing the concentration of NBT in thehas since improved with the adoption of synthetically reaction mixture also reduced the error due to interferingcreated glycated polylysine as a calibrator in new reducing substances other than uric acid.109 Thus thefructosamine kits. Reaction of this compound with NBT new assay is significantly more robust than previously.produces an absorption spectrum more akin to human

Body Mass Indexserum than that found with DMF, thereby, it is hoped,An unusual but consistent finding has been observedresolving most fructosamine calibration problems.77

whereby obese diabetic patients and non-diabetic individ-uals have been found to have lower fructosamine valuesSerum Proteins

Debate continues as to whether fructosamine concen- than lean ones.119–121 Reductions of up to 25 % havebeen noted in subjects with a body mass indextration should be corrected for serum protein concen-

tration. Empirically, it would not seem surprising that a (BMI) . 30 kg m−2 despite having similar HbA1c andfasting glucose values.119,121 Serum from these patientsmeasurement of glycated proteins is apt to be influenced

by serum protein concentrations. Opponents of this view has also been found to glycate serum proteins at a slowerrate in vitro than serum from less obese individuals.121argue that, theoretically, glucose concentration is the

rate-limiting step in the glycation reaction because However, the precise mechanism for this phenomenonremains unknown.available protein lysine residues will always be vastly

in excess of reactive open-chain (carbonyl) glucosemolecules.110 However, much evidence has accumulated Monitoring of Blood Glucoseto suggest this is unlikely to be the case. An in vitrostudy has actually found that the amount of fructosamine Clinical Utilityformed is in first-order relation to albumin concentrationand not glucose.111 The authors of the study postulated Until the early 1980s, self-monitoring of glycaemic

control for most diabetic patients was limited to thethat as carbonyl glucoses were removed by glycation,more glucose molecules isomerized to the open-chain measurement of glucose concentrations in urine samples.

Many patients found this method to be insensitive,form to maintain equilibrium.

824 E.S. KILPATRICK


REVIEWTable 4. Possible causes of inaccurate homeparticularly due to the problems associated with the renalblood glucose measurementthreshold for glucose. Indeed, at the time, adjustment of

insulin dosages according to urine tests was likened toUser errors Sample size

‘driving a car on roads with a 30 mph limit with a Sample placementspeedometer that only begins to work at greater than Test timing

Test strip storage70 mph’.122 The measurement by patients of blood ratherGlucose contamination bythan urine glucose was perceived as a means ofunwashed handssurmounting these problems.Falsification of results

Although for many years the accurate and precisemeasurement of blood glucose had been achievable in Analytical Sample haematocrit

errors Interfering substances:the laboratory setting,123 the technology for portableascorbateinstrumentation did not become available until the earlyparacetamol1970s. The first advance was in the development of ‘dry’salicylate

chemistry techniques which obviated the need for liquid uratereagents in glucose measurement. With this approach, creatinine

Sample oxygen tensionthe blood sample itself was the solvent in which theSample haemolysischemical reaction took place. The earliest demonstration

of such technology for glucose measurement occurredMagnitude of analytical errors vary depending onover 30 years ago using glucose oxidase ‘Dextrostix’ the meter used.

strips from Ames which were similar in principle tothose in use today.124,125 Reflectance meters were thenintroduced which gave the promise of more accurate and that for diagnosis, move away from laboratories into the

hands of diabetic patients and healthcare workers. Theconsistent measurement compared with visual reading.126

However, these first meters were bulky, expensive and use of meters has now become commonplace not onlyamong diabetic patients in the community but also fornot easy to use. For example, the Ames ‘Eyetone’

meter cost around £200, weighed 1.7 kg, measured the monitoring of acutely ill hospital patients. The latteris illustrated by the fact that in the author’s hospital trust,18 × 11 × 5 cm, required mains operation, a 30 minute

warm-up period, calibration before each sample, and over three ward glucose measurements are performedfor every sample sent to the biochemistry laboratory.employed a needle scale for reading.127

Despite these disadvantages, in 1978 a number of The next major advance in blood glucose monitoringis likely to be in the form of non-invasive measurementinvestigators demonstrated the potential usefulness of the

devices when used at home by diabetic patients.122,128,129 using near infra-red techniques132 but as yet no commer-cial system has been successfully developed.It was hoped that the ability to measure near physiological

blood glucose concentrations would allow more diabeticpatients to achieve this degree of control.122 It also meant Factors Influencing Blood Glucose Testthat glucose concentrations could be assessed accurately Strip Measurementwithout recourse to frequent hospital admission,especially in pregnant diabetic patients.128,130 Lack of Standardization

While the use of whole blood in a clinical setting hasNevertheless, it was recognized that the potentialmarket for meters would only be realized if they became the advantage of being convenient, whole blood glucose

measurement, unlike plasma glucose, lacks a definedless expensive and genuinely portable. Thus, therefollowed a period of steady improvement in the products standard against which other methods can be cali-

brated.133 However, the Yellow Springs Instrument (YSI)of all manufacturers in terms of ease of use and cost. In1987, the ExacTech meter from Medisense was particulary glucose analyser has become a de facto standard used

by most glucose meter manufacturers to calibrate theirgroundbreaking. It used a pen sized meter which, insteadof detecting a colour change in a reagent strip, employed instruments. The choice of the YSI owes more to it being

one of the first widely available whole blood glucosea disposable biosensor for the electrochemical detectionof blood glucose.131 It was also in the vanguard of instruments than to any analytical superiority. Indeed,

because the YSI measures blood glucose indirectly (i.e.introducing products which did not require the user towipe blood from the test strip during measurement. after dilution of the sample), results are liable to be

affected by deviations in plasma protein concentrations,Most new glucose meters are now compact and ‘non-wipe’ and several also include features such as automatic high lipid levels, and the haematocrit of the blood

specimens.133 Most reflectance meters in fact measuretiming of the test, time/date stamping of results, and therecording of these results in the meter memory. Figure 1 plasma glucose because the red cells are retarded from

entering the reagent layer of the test strip, but becauseillustrates a variety of new technology instrumentscurrently available in the UK. the meter is calibrated against the YSI it gives a whole

blood equivalent result. Boehringer Mannheim haveThe ensuing success of glucose test strips and metershas seen the measurement of blood glucose, other than broken with tradition by calibrating their Advantage



REVIEW

Figure 1. A selection of ‘new technology’ blood glucose meters. Top (L to R): Boehringer Mannheim Advantage; Bayer Glucometer4; Lifescan One Touch II. Bottom: Medisense meter.

meter against a whole blood hexokinase reference fabricated results, in 8 % results had been omitted andin 26 % there was disagreement between the meter resultinstrument rather than the YSI. However, it means results

from this meter tend to be more comparable to plasma and that written in the diary.140 The authors concludedthat about a third of patients could not be relied onvalues (i.e. higher) than if calibrated to the Yellow

Springs Instrument. Thus, the lack of an agreed standard, to keep unambiguous diaries, a figure confirmed insubsequent studies.141and the limitations of the adopted YSI instrument means

that whole blood glucose measurements cannot be asAnalytical Errors

universally concordant as that of plasma.Analytical compromises are unavoidable when using

a glucose meter instrument costing £30–50 and test stripsUser ErrorsAlthough newer glucose meters have helped address priced at £0.25 each. Indeed, it is perhaps surprising

how accurate and precise these meters can be consideringspecific problems, many errors in test strip glucosemeasurement are still attributable to the user. These such cost restraints. Nevertheless, some of the compro-

mises present can lead to clinically important errors ininclude errors due to the size and placement of theblood sample, the timing of the test and wiping of blood glucose measurement, especially under the wide range

of physiological conditions commonly found in hospitalfrom the test strip.134 Any other conceivable mistake inmeasurement such as the use of glucose-contaminated patients. Errors include those listed below and summar-

ized in Table 4.hands or inappropriate storage of test strips is sure tobecome apparent when any group of diabetic patients Sample Haematocrit. Variations in blood samples haema-

tocrit have been shown to be a source of inaccuracy inor health care staff are allowed use of the analysis.As reviewed previously in Diabetic Medicine,135 visual many blood glucose systems.142–145 In general, blood

samples with low haematocrits can give spuriously highreading of test strips adds another source of error whichcan considerably reduce the accuracy of measurements glucose results and vice versa. This effect can be well

in excess of that found when using instruments such asin general use.134 Patients tend to cluster their resultsaround the standard colours on the container, despite the YSI analyser. For example, for every 10 % change

in sample haematocrit the glucose concentration meas-instructions to interpolate.136 Colour blindness andimpaired colour discrimination, either inherited or ured by Bayer Glucostix can vary by approximately

20 %.142 Newer instruments such as the Bayer Gluco-acquired as a result of diabetes,137 can also lead todifficulty in strip interpretation.138,139 meter 4 and Boehringer Mannheim Accutrend have

effectively addressed this problem, but other contempor-Another important source of user ‘error’ is the falsifi-cation by patients of results obtained by glucose self- ary instruments, such as the Boehringer Mannheim

Advantage, the Medisense meter, and (to a lesser extent)monitoring. In the first study to highlight the problem,19 diabetic patients were given meters which had the Lifescan One Touch II still remain affected.146–150

Interfering Substances. When the glucose oxidase(unknown to them) memory chips built in. It was foundthat in 30 % of glucose record diaries the patient had enzyme is used for glucose measurement, reducing

826 E.S. KILPATRICK


REVIEWagents found in the blood sample can interfere by a and test strip blood glucose measurement, many clinically

relevant problems remain when assessing glycaemiccompetitive effect on the redox detection system.151

Hence, exogenously given substances such as ascorbic control by these routinely used means. Thus, althoughtechnological advances have led to improvements,acid, paracetamol and salicylate, as well as large amounts

of endogenous uric acid, urea and creatinine can lead interpretation of these indices cannot yet be used as asole substitute for clinical judgement.to falsely low results with many systems.151 Recent

products have approached this problem by using teststrips with alternative enzymes to the glucose oxidase

Referencessystems. For example, the Glucometer 4 meter fromBayer uses hexokinase while the Advantage meter from

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Problems in the assessment of glycaemic control in diabetes mellitus

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