HbA1c:Review of Standardisation and Quality Management

Cas Weykamp

(Queen Beatrix Hospital，Winterswijk Netherlands)

1 Introduction

Diabetes is characterised by chronic hyperglycaemia and long-term exposure to elevated glucose levels accelerates the progression of macro-and micro vascular diseases leading to the well-known longterm complications retinopathy，neuropathy，nephropathy，and cardiac diseases.Due to changes in life-style-we eat more and different，we exercise less there is an unbalance in energy intake and use，initially leading to overweight，later frequently followed by diabetes type 2.Diabetes has become a real global epidemic.Some numbers:346 million people have been diagnosed worldwide and in US 1 of 7 dollars of the health budget is diabetes related［1-2］.Efficient and effective management is required to handle this huge number of chronically diseased persons.Portable glucose meters facilitate short-time management.Long-term prospective studies，notably the Diabetes Control and Complications Trial(DCCT)，the United Kingdom Prospective Diabetes Study(UKPDS)and the Epidemiology of Diabetes Interventions and Complications Study (EDIC)，provided the evidence of the direct relation between diabetic complications and mean glycemia measured by glycated haemoglobin A1c(HbA1c)［3-5］.But to make HbA1cthe ultimate tool to monitor long-term glycemic control with specific targets for treatment and decision limits for diagnosis and screening，2 issues have to be addressed:strict standardisation and stringent quality management of the test.This review describes the history and present status of standardisation and the aspects of good quality management.

2 Standardisation

The need for standardisation derives from the success of HbA1c:the diagnostic industry well recognised the importance of(and the market for)HbA1c.Many methods in many more commercial versions have been developed.All have their own specificities and，unfortunately，their own reference ranges.This broad variation in test-results raised the need for standardisation［6］.

2.1 The boom of methods

There are 2 mainstream analytical concepts，one based on the separation of haemoglobin fractions，the other on specific chemical reactions(Figure 1).HbA1cand non-glycated haemoglobin(A0)have different electrochemical properties，which allow separation of the respective fractions and quantification of HbA1cas fraction of total haemoglobin.This concept is applied with ion exchange chromatography，capillary electrophoresis，and affinity chromatography.Due to the attachment of glucose to the β-valine terminal，the isoelectric point of HbA1cdiffers 0.02 pI units from A0.The difference is enough to allow separation with ion exchange chromatography but also that small that only dedicated high performance liquid chromatography (HPLC)instruments perform satisfactory［6］.Capillary electrophoresis also uses differences in charge.A high voltage(10.000 volt)electrical field and electro osmotic flow force separation［7］.Haemoglobin molecules with a glucose attached have affinity to boronic acid.In a column filled with boronic acid coated particles，HbA1cwill be delayed while HbA0runs freely through the column.This results in 2 fractions，glycated and non-glycated，and is the principle of affinity chromatography［8］. Chemical methods require 2 independent assays，one for HbA1cand one for total haemoglobin.HbA1cis measured using a specific chemical reaction.In immuno assays an excess of anti-HbA1cantibodies is added to a sample.Antibodies bind to HbA1cand their excess is agglutinated.The resulting immune complexes lead to cloudiness，which is measured turbidimetrically or nefelometrically［9］.In enzymatic assays a protease is used to cleave the β-chain.The resulting peptide reacts with an oxidase and the HbA1cconcentration is measured by determining the resulting hydrogen peroxide.In both immunochemical and enzymatic assays，total haemoglobin is measured photometrically［10］.

2.2 History:harmonisation efforts

Specificities and selectivities of commercial methods are different and therefore there is a broad variation in(uncalibrated)outcome of HbA1ctests.The first years after the discovery of HbA1c，each method or even each laboratory had its own reference values.For optimal clinical use (uniform clinical guidelines，comparison of scientific studies)equivalence of results is desirable.Equivalence can be achieved with harmonization or standardization［11］.With harmonization commercial methods are calibrated against an arbitrary chosen so called designated comparison method.With standardization calibration is against a scientifically sound reference method，a so called reference measurement procedure.One could also say that harmonization leads to a relative truth and standardization to the absolute truth.The need for equivalent results was well recognized and inspired several national harmonization initiatives.In the US the arbitrary chosen method was the method used in the DCCT and a nationwide program with international affiliations was(and is)organized by the NGSP［12］.Similar initiatives achieved harmonization in Japan(JDS/JSCC)and Sweden (Mono-S)［13-14］.Unfortunately all were based on designated comparison methods and it is not surprising that results of these arbitrary chosen methods were different.This situation caused confusion and therefore the IFCC developed a reference method to achieve worldwide standardization.

Figure 1 Analytical Concepts of HbA1c Methods and Their Traceability to the IFCC-RMP

2.3 Standardisation:IFCC Reference Measurement Procedure

The IFCC Reference Measurement Procedure(IFCC-RMP)is based on the concept of metrological traceability(Figure 2).Pure HbA1cand HbA0are mixed to prepare primary calibrators that are used to calibrate the IFCC-RMP.Erythrocytes are washed and lysed，followed by enzymatic cleavage［14-15］.The resulting hexapeptides are quantified with either HPLC-mass spectrometry or HPLC-capillary electrophoresis.With the IFCCRMP values are assigned to whole blood panels that serve as secondary calibrators for manufacturers.The IFCC-RMP is embedded in global network of reference laboratories［16］.The Shanghai Center for Clinical Laboratory(SCCL)was the first approved chinese laboratory in the global network.

Figure 2 The Metrologic Traceability Chain for HbA1c

Figure 3 shows the complete quality chain.With the IFCC-RMP values are assigned to the IFCC calibrators.These are used by the manufacturers to assign values to their kit calibrators，subsequently used by the clinical laboratories to calibrate their instruments.This chain warrants that worldwide HbA1cresults reported to diabetologists and patients are traceable to the IFCCreference method，allowing global guidelines with uniform decision limits for diagnosis and therapy.Independent control is achieved by external quality assessment/proficiency testing(EQA/PT)organizers using samples to which values have also been assigned with the IFCCRMP.

Figure 3 Quality Chain of IFCC-RMP Standardised HbA1c Testing

2.4 Dilemma:reporting units

In the medical laboratory it is common that，once a reference method has been established，patient results are expressed in the units of that reference method.In case of HbA1cchemists adopted the units of the IFCC-RMP but in some parts of the world(especially US)there was resistance of clinicians:they were used to the NGSP units and did not want to change.This was a dilemma solved in a summit meeting of IFCC，IDF，EASD and ADA:the IFCCRMP is the only valid anchor for standardization of HbA1cbut on patient reports HbA1cwill be reported in both IFCC(mmol/mol)and NGSP(%)units［17］.NGSP units are derived from IFCC units using a master equation.Reporting in two units is not practical in daily life and therefore many countries use either IFCC or NGSP units.Table 1 shows standard interpreation norms in both units.Instruments have both options and journals follow the consensus statement and publish parallel in both units.The master equations to convert IFCC into NGSP-units［NGSP(%)=0.0915X IFCC(mmol/mol)+2.15］and vice versa［IFCC(mmol/mol)=10.93 NGSP(%)－23.5］are established and monitored by the networks of IFCC and NGSP［16］.

Table 1 Standard Interpretation Norms

3 Quality management

HbA1cis the ultimate longitudinal parameter:therapy of a patient depends on serial measurements of HbA1cover a period of years or even decades.For this reason quality management should be given much attention.Aspects are pre-and post-analytical considerations，the quality system，and performance goals.Point-of-care tests(POCT)，operated outside the laboratory should be given specific attention.

3.1 Pre-analytical considerations

Contrary to glucose，the sample collection and storage of HbA1cis robust.Blood can be taken any moment of the day without any precautions of the patient.Blood obtained by venipuncture or fingerstick capillary is suitable.Unless otherwise specified by the manufacturer the anticoagulant should be ethylene diamine tetraacetic acid(EDTA).Sample stability is method specific with methods based on differences in charge being most sensitive to ageing effects on the results.Some POCT cannot measure when the specimen is even only slightly haemolysed.But in general blood is stable up to 1 week in the refrigerator(2-8°C).Frozen below－70 °C blood is stable for at least a year.It should be stressed that storage at－20 °Chas adverse effects and should be avoided［20］.Some manufacturers developed methodspecific collection systems to facilitate the logistics after field-collection(e.g.filter paper and micro cups with lysing buffer).These systems should only be used if validated in comparison with standard collection［21］.

3.2 Post-analytical considerations

Results below the lower limit of the reference interval should be confirmed with repeated testing.If confirmed，the clinician should be informed on the possibility of a variant or shortened red cell survival.Samples with extreme high results(＞140 mmol/mol;＞15%)and results that do not match with the clinical picture should also be re-assayed.Repeated testing with a method of a different analytical principle can be of help to trace the reason for unexpected results(patient-or method related).

3.3 Quality system

In qualified laboratories a quality system consisting of three basic ingredients is in place:accreditation to ISO 15189，internal quality control and external quality control. For internal quality control， 2 control materials(low and high HbA1c)should be assayed in every analytical run with results within pre-defined limits.Frozen whole blood aliquots stored below－70℃and lyophilised haemolysates with no or known matrix effects are suitable［21］.Participation in an EQA (also:PT)provides valuable external information to manage the quality of HbA1c:bias is derived from the EQA target set with the IFCC-RMP，performance is compared with other laboratories using the same method and EQA reports are an up to date review of available methods and their performances.

3.4 Performance goals

The reliability of an HbA1cresult depends on bias(related to proper calibration)and precision(related to the reproducibility of the method).Quality goals can be derived from biological variation，clinical needs or the state of the art.For HbA1ca generally accepted rule of thumb is that clinicians interpret a difference of 5 mmol/mol(0.5%)between successive patient samples as a significant change in glycemic control［22］.From this clinical need，it can be calculated that the intralaboratory CV(derived from the lab's internal quality control records)should be＜3%(＜2%NGSP units).The interlaboratory CV(derived from the EQA review)should be overall＜5%(＜3.5%NGSP units)or＜4.5%(＜3%NGSP units)within one method［21］.

3.5 POCT

The quality concept used in laboratories can not be applied in POCT settings.Careful reading of the instructions，check if the manufacturer warrants traceability of results to the IFCC-RMP，and periodic exchange of samples with a qualified laboratory contribute to the quality.

4 Future perspective

Due to the IFCC and NGSP standardisation and ongoing efforts of the diagnostic industry，HbA1chas much improved since the landmark clinical trials(DCCT，UKPDS)clearly demonstrated the relationship between glycemic control，HbA1cand diabetic complications.HbA1cis now a reliable test and as such an indispensable tool in both routine management and diagnosis of diabetes.But there are still things to do.Global availability of adequate methods，traceable to the IFCC-RMP has not been achieved yet.Although the IFCC-RMP has been adopted as the only valid anchor to standardise，HbA1cis still reported in several different units:universal reporting in IFCC-units remains a challenge.Quality management in laboratories needs to be improved.The use of POCT instruments is controversial.Only a few devises meet acceptable performance criteria，even in the hands of laboratory professionals［23］.The quality of testing by nonlaboratories is questionable:as long as participation in an EQA programme is not mandatory，no objective information on performance in the real world is available.Improvement of the tests and development of a quality concept to warrant acceptable performance by non-laboratory staff is a challenge.And finally，education，education:for the management of diabetes more and better tools are available now，but there is still much to be learned for laboratories and clinicians on standardisation，quality management and how to use HbA1cto achieve the best patient care.

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