HbA1c — An analyte of increasing importance

Clinical Biochemistry 45 (2012) 1038–1045

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Review

HbA1c — An analyte of increasing importance Trefor Higgins ⁎ DynaLIFEDX, Edmonton, Alberta, Canada, T5J 5E2

a r t i c l e

i n f o

Article history: Received 6 March 2012 Received in revised form 4 June 2012 Accepted 6 June 2012 Available online 14 June 2012 Keywords: HbA1c Glycated hemoglobin Diabetes mellitus

a b s t r a c t Since the incorporation in 1976 of HbA1c into a monitoring program of individuals with diabetes, this test has become the gold standard for assessment of glycemic control. Analytical methods have steadily improved in the past two decades, largely through the efforts of the National Glycohemoglobin Standardization program (NGSP). The new definition of HbA1c and the introduction of an analytically pure calibrator have increased the possibility for greater improvements in analytical performance. Controversies exist in the reporting of HbA1c. The use of HbA1c has expanded beyond the use solely as a measure of glycemic control into a test for screening and diagnosing diabetes. With improvements in analytical performance, the effects of demographic factors such as age and ethnicity and clinical factors such as iron deficiency have been observed. In this review, the history, formation, analytical methods and parameters that affect HbA1c analysis are discussed. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Contents History of HbA1c . . . . . . . . . . . . . . . . . . . . . . . . Discovery . . . . . . . . . . . . . . . . . . . . . . . . . Formation . . . . . . . . . . . . . . . . . . . . . . . . . Definition of HbA1c . . . . . . . . . . . . . . . . . . . . . HbA1c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reference method . . . . . . . . . . . . . . . . . . . . . Repercussions from the new definition/calibrator . . . . . . Analytical methods . . . . . . . . . . . . . . . . . . . . Use of HbA1c . . . . . . . . . . . . . . . . . . . . . . . . . . Assessment of glycemic control . . . . . . . . . . . . . . . Diagnosis of diabetes . . . . . . . . . . . . . . . . . . . . Assessment of risk to progression to diabetes in non-diabetics Assessment of cardiovascular risk in non-diabetics . . . . . . Factors influencing HbA1c results . . . . . . . . . . . . . . Analytical factors . . . . . . . . . . . . . . . . . . . . . Demographic factors . . . . . . . . . . . . . . . . . . . . . . Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gender . . . . . . . . . . . . . . . . . . . . . . . . . . Ethnicity . . . . . . . . . . . . . . . . . . . . . . . . . Seasonality . . . . . . . . . . . . . . . . . . . . . . . . Clinical . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glycation phenotype . . . . . . . . . . . . . . . . . . . . Smoking . . . . . . . . . . . . . . . . . . . . . . . . . Iron deficiency . . . . . . . . . . . . . . . . . . . . . . . Effects of drugs . . . . . . . . . . . . . . . . . . . . . . Future directions and predictions for HbA1c . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Fax: + 1 780 452 2845. E-mail address: [email protected]. 0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2012.06.006

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T. Higgins / Clinical Biochemistry 45 (2012) 1038–1045

History of HbA1c Discovery In 1958 Huisman and Mayering [1], using micro-columns, separated hemoglobin into several fractions, labeling them HbA0, HbA1 and HbA2. Allen [2] further resolved HbA1 into 3 fractions, naming them “fast hemoglobins” or HbA1a, HbA1b and HbA1c. Further investigation resolved HbA into 2 components, namely HbA1a1 and HbAa2. HbA1a1 was found to be the glycation product at the N-terminal of hemoglobin with fructose 1, 6 diphosphate and HbA1a2 the glycation product of the N-terminal of hemoglobin with glucose 6 phosphate. Although there is not unanimity on the identity HbA1b, it is commonly described as pyruvic acid attached to the N-terminal of hemoglobin. HbA1a and HbA1c decrease with improved glycemic control [3]. However, it was not until 1968 that Brookchin and Gallop [4] established that HbA1c was a glycoprotein. Rahbar et al. [5] discovered in 1969 that HbA1c was increased in the blood of patients with diabetes. Bunn et al. [6] in1975 described the 2 phase formation of HbA1c. In 1976, Koening, Cerami and their coworkers [7] included HbA1c in the monitoring of glucose metabolism and pioneered the clinical utility of HbA1c in monitoring glycemic control in patients with diabetes mellitus. Formation HbA1c formation as shown in Fig. 1, is a 2 stage non-enzymatic process. In the first step, a fast step, glucose attaches to the N-terminal of hemoglobin to form an unstable aldimine intermediate (Schiff's base). This intermediate either undergoes an Amadori rearrangement to form, in a slow step, a stable ketoamine, HbA1c, or reverts to glucose and hemoglobin. From this equation it follows that the concentration of HbA1c is related to the glucose concentration [8–10], and since the average life span of the red blood cell is some 120 days, HbA1c is viewed as the average blood glucose over the past 120 days. However, the test is more reflective of the glucose concentration in the past 28 days rather than the past 120 days [11,12]. Definition of HbA1c Based on the European Union directive (EU ISO 17111 2003) for in vitro diagnostic medical devices, it was necessary that HbA1c should be better defined than a glycoprotein band on a column and the measurement system be metrologically traceable. The International Federation of Clinical Chemistry (IFCC) set up a Working Group that first developed a definition for HbA1c [13] and then, based on the definition, produced a calibrator [14] and then established a reference

HbA-Val-NH2

HbA-Val-N

HbA-Val-NH

+ HCO

HC

CH2

HCOH

HCOH

C=0

HOCH

HOCH

HOCH Amadori rearrangement

HOCH

HOCH

HOCH

HOCH

HOCH

HOCH

CH2OH

CH2OH

CH2OH

Glucose

Unstable Schiff's base

Ketoamine

HbA

Labile Fig. 1. Formation of HbA1c.

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method [15,16] to accurately quantify HbA1c in blood. The new definition was that HbA1c is the “the substance fraction of the β chains of hemoglobin that has a stable hexose adduct on the N-terminal amino acid valine “or β N-1-deoxyfructosy-hemoglobin. This definition eliminated glucose adducts to the lysine amino acids in the β globin chains and the α globin chains of hemoglobin from the measurement of HbA1c. HbA1c Reference method The reference method for measuring HbA1c involves the incubation of blood with the endoproteinase Glc-C, resulting in cleavage of the Nterminal hexapeptide of the β chain with subsequent separation and quantification of the glycated and nonglycated hexapeptides by mass spectroscopy [15–17]. It has been recommended that all HbA1c methods be based (anchored) on this IFCC HbA1c calibrator [18]. Repercussions from the new definition/calibrator The elimination of adducts from HbA1c quantification resulting from this new IFCC reference calibrator made HbA1c results using this calibrator consistently lower by about 2% than [17] the National Glycohemoglobin Standardization Program (NGSP) which was the major calibration system used worldwide. HbA1c values derived using the IFCC standard also differed from calibration systems used in Japan (Japanese Diabetes Society, JDS) and Sweden (Mono S) [17]. To avoid potentially difficult situations where laboratories in an area would use two different HbA1c calibration systems but the same unit of measurement, the IFCC proposed the use of SI numbers (mmoll/mol) for HbA1c measurements using the IFCC calibrator. The International HbA1c Consensus Committee, comprised of representatives of the American Diabetes Association (ADA), the European Association for the Study of Diabetes (ESAD), the IFCC and the International Society for Pediatric and Adolescence Diabetes (ISPAD), issued a consensus statement on the worldwide standardization of HbA1c. In this statement it was recommended that HbA1c be reported in both SI units [18] and percentage units (NGSP). Also recommended was the reporting of estimated Average Blood Glucose (eAG) on the grounds that patients were familiar with glucose values [19,20] but had difficulty converting HbA1c values into the glucose values obtained on their Self Monitoring of Blood Glucose (SMBG) program. The reporting of eAG [21] was based on a study involving over 600 subjects in 10 international centres and has been criticized as having too limited enrollment as results from some sites were not included in the study due to analytical problems. The inclusion of eAG along with HbA1c is controversial [22–24] and has not been universally adopted [25,26]. The underlying challenge to the use of the reporting of estimated average glucose is the variability in the relationship of glucose to HbA1c [10]. Analytical methods Analytical methods for HbA1c quantification may be classified either by the analytical principle on which the analysis is based or the location where the test is performed. The two analytical principles, on which HbA1c assays are based, together with examples of the testing methods, are: 1) Methods based on charge differences (HPLC, electrophoresis). 2) Methods based on structural differences (immunoassay, boronate affinity chromatography, enzymatic). Alternatively, HbA1c methods may be classified on location of testing:

Stable as HbA1c

1) Central laboratory testing (immunoassay, HPLC, boronate chromatography enzymatic).

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2) Point-of-Care (POC)(boronate affinity). 3) Large Clinic testing (boronate affinity, immunoassay). The preferred sample for HbA1c analysis is EDTA anti-coagulated blood. HbA1c is a relatively stable analyte with a study showing that exposure of samples to temperatures of ≥ ≤ 30° should be avoided and, that for HPLC methods, storage at − 4° is better than storage at − 20°. When long-term storage is needed, samples should be stored at − 70° [27]. Degradation products are noted on HPLC analysis 3 to 4 days after collection and may or may not interfere with the measurement of HbA1c. Carbamylated hemoglobin, the product of urea attaching to the N- terminal of hemoglobin and found in relatively high concentrations in patients with renal disease, used to interfere with HPLC methods for HbA1c [28] but this is no longer the case. At one stage the unstable Schiff base (labile fraction) interfered with HbA1c analysis and so recent recommendations [29] now advocate the elimination of this interference. Initially this removal was achieved by incubation of the sample with different buffers at 37°. This interference is not an issue with immunoassay methods due to the specificity of the antibody; in HPLC methods it is resolved either by separating the labile fraction from the HbA1c or by complex mathematical algorithms that eliminate any potential interference. The transportation of whole blood samples used for measuring HbA1c in population studies may present some challenges and the use of dried filter spot samples has been determined to be robust, accurate and reproducible, providing the collection method is standardized [30,31]. Independent of the analytical principle, type of sample or location of testing, it is recommended that the analytical method used be certified by the National Glycohemoglobin Standardization program (NGSP) and meet the analytical specifications outlined in the 2011 National Academy of Clinical Biochemistry Guidelines (NACB) [29]. In brief, these recommend that the analytical intra-laboratory CV should be b2.0% and the inter-laboratory CV be b 3.0%. Other groups have made similar recommendations with similar values [32,33]. It is suggested that the analytical goals for HbA1c measurements using NGSP and IFCC units are different with an outcome-based analytical goal of 0.5% ( unit of measurement) or 7.9% (percentage) using the NGSP calibration system and 5.0 (unit of measurement) or 8.65% (percentage) using the IFCC calibration system [34]. Participation in a proficiency testing program is also recommended [29] as is confirmation testing when the HbA1c value is above 15% or below the reference interval of the method used [29]. Since POC tests are waived by the United States Federal Food and Drug administration (FDA), many laboratories using POC methods and some suppliers of POC materials do not subscribe to the NGSP certification program and the analytical performance of these methods is less than optimal [35–40]. The analytical performance of HbA1c testing has improved since 1996 when the National Glycohemoglobin Standardization Program (NGSP) was introduced [41]. This improvement is observed as decreased variability between methods and improved precision within methods. A quality assurance program for HbA1c testing using HPLC as the testing method has been suggested [42]. The biological variation of HbA1c in non-diabetics is low [43–46] and is usually considered as b2.0%. If the analytical performance of a HbA1c method in a laboratory is the recommended b2% and the biological variation is 2% then the critical difference is 7.2%. At the glycemic control target of 7% the critical difference becomes 0.5%, meaning that if an initial HbA1c value of 7% was initially obtained in a patient then a subsequent HbA1c value may fall between 6.5% and 7.5% due solely to analytical and biological variation. In a study performed in our laboratory over 90% of HbA1c results performed either 3 or 6 months after the initial test differed by less than 0.5%.

Skeie and co-workers based desirable analytical performance for HbA1c methods on patient and physician expectations [47,48]. They found that patients were more demanding than their physicians, requiring an analytical CV of 3% to meet their expectations. It is recommended that the reference range for HbA1c for NGSPcertified methods “should not deviate substantially (e.g. >0.5%) from 4% to 6% [29]. However, a study showed that there were substantial differences in reported HbA1c reference ranges among laboratories in the United States, despite all the methods used in these laboratories having NGSP certification [49]. The term HbA1c has being recommended [18] as the term to describe the glycated product of hemoglobin. The Canadian and American Diabetes Associations recommend the term A1C on the grounds that it is shorter and patients may have confusion as to why hemoglobin is measured when they have diabetes. Although purists may argue that HbA1c is the best scientific term to describe the glycated product of hemoglobin the term HbA1c is close to the true scientific term and eliminates the need to subscript. The term A1c seems to have very little acceptance among laboratory groups. Use of HbA1c Assessment of glycemic control The Diabetes Complication and Control Trial (DCCT) in individuals with Type 1 diabetes mellitus established that intensive insulin therapy produced good glycemic control, as measured by HbA1c, and significantly reduced the complications of diabetes when compared to conventional insulin therapy [50]. A reduction in HbA1c level caused a decrease in the incidence of the complications of diabetes mellitus namely, retinopathy, neuropathy and nephropathy - with optimal reduction achieved at a HbA1c value of 7%,. A study performed in the United Kingdom in patients with Type 2 diabetes (United Kingdom Prevention Diabetes Study (UKPDS) showed similar results to the DCCT trial in that good glycemic control, as measured by HbA1c, resulted in a reduction of diabetes-induced complications [51]. Based on these studies, HbA1c testing may be used to monitor the effectiveness of therapy or patient compliance to therapy within an individual. Measuring HbA1c across individuals within a therapy program provides a measure of the effectiveness of the program. Both the American Diabetes Association (ADA) [52] and the Canadian Diabetes Association (CDA) [53] recommend a target treatment goal of 7% for individuals with Type 1 or Type 2 diabetes to reduce microvascular and macrovascular complications in these patients. The CDA guidelines suggest a target HbA1c of 6.5% be considered in individuals with Type 2 diabetes to reduce the risk of nephropathy, provided consideration is given to the risk of hypoglycemia [51]. The CDA guidelines also recommend a target HbA1c value of 8.5% in children under 5, b8.0% in children 6 to 12 and ≤7.0% in children 13 to 18 years of age. For pre-pregnancy, the CDA recommends a target HbA1c of ≤7.0% and, during pregnancy, a HbA1c of ≤6.0% (or within the reference range) is recommended. The American Diabetes Association (ADA) [52], Canadian Diabetes Association (CDA)[53] and the UK National Institute for Health and Clinical Excellence (NICE) [54,55] provide guidance for testing frequency. Both the CDA and ADA recommend HbA1c testing every 3 months in patients who have poor glycemic control or where there is a change in therapy and every six months in patients who have good glycemic control and have not had a change in therapy. The UK NICE guidelines recommend that HbA1c be tested every 2 to 6 months in patients with unstable diabetes and that a measurement made at an interval of less than 3 months be used as an indication of change rather than steady state. For patients with well-controlled diabetes, the time interval between testing is recommended to be 6 to 12 months. These guidelines are reinforced by the National Health System (NHS) Clinical Knowledge Summaries. Studies [56] have

T. Higgins / Clinical Biochemistry 45 (2012) 1038–1045

shown that compliance with this type of recommendation is poor [57–60]. Diagnosis of diabetes Although HbA1c was not originally recommended for the diagnosis of diabetes, an Expert Committee in 2006 recommended that HbA1c be used in the diagnosis of diabetes mellitus using a threshold value of 6.0% [61]. In 2009 an Expert Committee, using data from the National Health and Nutrition Examination Study (NHANES) III, recommended a threshold value of 6.5% for the diagnosis of diabetes mellitus [62]. This recommendation has been adopted by the American Diabetes Association (ADA) [63] and the American Association of Endocrinologists in 2010 [64], and the World Health Organization [65] and the Canadian Diabetes Association (CDA) in 2011 [66]. The addition of HbA1c in the criteria for the diagnosis of diabetes mellitus has been controversial with articles advocating its use [67,68] and those repudiating its use [44,69,70]. Olson et al. in particular concluded that the proposed criteria advocated by the ADA were insensitive [70]. In general, advocates for the use of HbA1c as a screening test for diabetes point out [67]: 1) HbA1c requires no patient preparation as opposed to the fasting requirements for fasting blood glucose collection. Patient compliance with the fasting requirement is questionable. Furthermore, the fasting glucose test must be confirmed on a separate occasion, increasing the possibility of patient non-compliance. 2) If an oral glucose tolerance test (OGTT) is used to diagnosis diabetes, the patient must fast for a minimum of 8 hours, ingest an unpleasant-tasting drink and wait, without leaving the collection location for 2 hours, for a second collection. Furthermore, a second confirmation glucose test is required and OGTT tests are irreproducible [71–73]. 3) The biological variation of glucose is higher than that of HbA1c (5.1v1.9%). 4) There are no pre-analytical challenges associated with HbA1c tests but many associated with a glucose test [42,74,75]. 5) The HbA1c is well-standardized with equivalent results obtained by laboratories using methods certified by the NGSP. 6) HbA1c is more reflective of microvascular complications than glucose. Although these arguments have merit on the surface, it has been suggested that the use of HbA1c as a screening test for diabetes is based on patient convenience rather than strong evidence-based data [69]. The endorsement of the major diabetes associations in both North America and the World Health Organization ensures that the diagnosis of diabetes using HbA1c will be acceptable clinical practice.

Assessment of risk to progression to diabetes in non-diabetics In the Insulin Resistance Atherosclerosis Study (IRAS)[76], it was found that HbA1c values between 5.7 and 6.4% were not as good for detecting individuals at risk for diabetes compared to a fasting glucose in non-Hispanic whites. Olson et al. [70] found no use in detecting pre-diabetes using HbA1c. A Japanese study [77] as found that very few patients with an initial HbA1c value of ≤6.0% progressed to an HbA1c value of ≥6.5% within 3 years and recommended that, based on this data, screening for diabetes using HbA1c should not be performed at intervals of b3 years if the initial HbA1c was below 6%. Ginde et al. [78] developed a risk stratification strategy based on the patient's age, gender, ethnicity (Black ), hypertension, elevated waist circumference, elevated triglycerides and low HDL cholesterol. In the moderate and high risk groups, a threshold HbA1c value ≥6.1% identified patients requiring a fasting glucose to confirm the diagnosis of diabetes and an HbA1c ≤ 5.4% excluded a diagnosis of

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diabetes. An HbA1c between 5.5 and 6.0% may exclude diabetes in moderate risk groups but not in high risk groups. Despite the limitations of using HbA1c as a screening or diagnostic test for diabetes, it has been suggested that it may be a good rule-out test for these purposes [69]. Assessment of cardiovascular risk in non-diabetics The 10 year follow-up of the Hoorn study [79] found that, in individuals between 50 and 75 years of age without diabetes, HbA1c was the best predictorof 10 year fatal and non-fatal cardiovascular events and all-cause mortality, compared to fasting and 2 h post prandial glucose. In the North American Atherosclerosis Risk in Communities (ARIC) study, it was found that in non-diabetics HbA1c was a better predictor of diabetes and cardiovascular disease compared to fasting glucose [80]. Factors influencing HbA1c results There are several factors which influence HbA1c values and these may be categorized as: analytical (presence of hemoglobinopathies and/or HbF), demographic (age, gender, ethnicity and seasonality/ temperature change) or clinical (decreased red cell survival, smoking, iron deficiency, vitamin use, decreased/increased glycation of hemoglobin due to phenotype and biological variation). These are summarized in Table 1 [65]. Analytical factors Some HbA1c methods, such as HPLC, are more prone to interference than immunoassay methods. The interference may be noted as an absurd HbA1c value or an HbA1c value that is inconsistent with the glucose concentration. The 2011 NACB recommendations [29] recommend that a HbA1c value greater than 15% or below the lower limit of the reference range be repeated (preferably by a method based on a different analytical principle). In HPLC the HbA1c may be elevated due to the co-elution of the variant with HbA1c, producing an absurd result. For example, the co-elution of HbI with HbA1c produces HbA1c values in excess of 25% and the co-elution of HbA1c with Hb K Woolwich, Hb Hope and Hb Camden produces HbA1c values in excess of 45%. In these cases, clearly an alternative method, usually immunoassay, must be used for quantification of HbA1c. However when an immunoassay HbA1c method is used with these hemoglobin variants HbA1c values obtained are below 4% and are inappropriately low for the glucose concentration. Possibly the variant hemoglobin interferes with the attachment of the variant β cahin to the antibody. In some cases the variant co-elutes with HbA but the glycated species does not co-elute with HbA1c, producing a falsely low HbA1c value. Table 2, although not exhaustive, provides a list of hemoglobinopathies that are known to affect the HbA1c value by HPLC. The most common hemoglobin variants do not usually interfere with most methods [81–86] and the National Glycohemoglobin Standardization Program website contains up-to-date information on the interference of hemoglobin variants with different methods. In general, boronate affinity methods do not show interference from hemoglobin variants in the measurement but there is a report of hemoglobin Constant Spring interference [87]. Immunoassay methods are prone to interference from HbF [88] due to the fact that most antibodies used in quantification of HbA1c recognize the glycated first 5 or 6 N terminal amino acids of the β globin chain. In HbF there are changes in the amino acid composition in these positions and so the antibody does not recognize glycated HbF. However, the reagents used in the quantification of the total hemoglobin measure both HbA and HbF and so the resultant HbA1c value is falsely low.

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Table 1 Factors that can affect A1C (reference [66]). Factor

Increased A1C

Decreased A1C

Erythropoiesis

Iron deficiency B12 deficiency Decreased erythropoiesis

Use of erythropoietin, iron or B12 Reticulocytosis Chronic liver disease

Altered hemoglobin

Variable change in A1C

Fetal hemoglobin Hemoglobinopathies Methemoglobin Genetic determinants

Glycation

Alcoholism Chronic renal failure Decreased erythrocyte pH Increased erythrocyte lifespan: Splenectomy

Erythrocyte destruction

Assays

Hyperbilirubinemia Carbamylated hemoglobin Alcoholism Large doses of aspirin Chronic opiate use

Interference from a hemoglobin variant, HbS, and the presence of HbF has been reported [89] and this case illustrates the many challenges of reporting HbA1c in the presence of hemoglobin variants and HbF. It is suggested that the presence (or preferably, the identity) of a hemoglobin variant found on analysis for HbA1c be reported [90]. Demographic factors Age It appears logical that if plasma glucose increases with age [29], then HbA1c would also increase with age. However, the literature on Table 2 Twenty-seven hemoglobin variants that interfere with measurement of glycated hemoglobin by ion-exchange chromatography (from color atlas of hemoglobin disorders. College of American Pathologists. 2003. Chicago). Variant

Reference

F* J K Bart's H N I Hope Raleigh South Florida Deer Lodge Okayama Marseille/Long Island Andrew-Minneapolis Osier Sherwood Forest Fukuyama Tatras Lisbon Malmo Tacoma Fannin-Lubbock Olomouc Hifiyama Le Lamentin Graz Hokusetsu

References to these variants may be found in textbooks.

Source listed in Elder et al. [128]

Ingestion of aspirin, vitamin C or vitamin E Hemoglobinopathies Increased erythrocyte pH Decreased erythrocyte lifespan: Chronic renal failure Hemoglobinopathies Splenomegaly Rheumatoid arthritis Antiretrovirals Ribavirin Dapsone Hypertriglyceridemia

Hemoglobinopathies

the effect of age on HbA1c values is divided, with the majority of reports indicating that there is an increase in HbA1c value with age [49,91–95] while one study shows no difference with age [96]. Gender Information on gender differences shows that the effect of differences in gender tends to be accentuated in adolescent females compared to males of the same age [97]. Dilli et al., on the other hand, found that boys and high fasting glucose levels were associated with high HbA1c but this association was not as strong in females [98]. Parikh et al. [99] found that in adult females increasing HbA1c values correlated better to increased all-cause and cardiovascular mortality than in men. Ethnicity There is no doubt that ethnicity is an important contributing factor HbA1c values and that is may be as much as 0.4% between Blacks and Caucasians. However some if this has clinical significance [29] to those who suggest that this difference is so significant that on set of threshold vales for of HbA1c values could be applied to different ethnic groups [100]. The consensus of the literature [49,101–104] is that Caucasians have the lowest HbA1c with Mexican Americans having higher HbA1c values and Blacks having the highest HbA1c among non-diabetic, impaired glucose tolerance and diabetic individuals. Seasonality There is significant evidence that HbA1c levels are higher in colder temperatures [105–112] compared to warmer temperatures, and that the larger the difference in temperature between summer and winter, the larger will be the difference in HbA1c between summer and winter. In Singapore where there is very little difference (0.1 degree) between summer and winter temperatures, Higgins et al. [112] found there was no variation in mean HbA1c values in the year. Others [113,114] have associated the changes in HbA1c levels during the year to holiday seasons and attribute the increase in HbA1c to the over-consumption of food during the holiday season. Hawkins [114] pointed out that between the start of December and the end of January there are many celebrations in the multi-cultural society

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of Singapore and all are celebrated independent of ethnicity or religious background. His data contradicts that of Higgins et al. [112]. Clinical Glycation phenotype Not all individuals are created equal regarding their ability to glycate hemoglobin. It is noticed by clinicians that some individuals have higher HbA1c levels with the same glycemic control, as measured by self-monitoring of blood glucose, and ethnicity than others. This change in level of HbA1c between individuals has been attributed to different phenotypes in people with higher than expected HbA1c, termed “fast or high” glycaters, while those with lower than expected HbA1c values are termed ”slow or low” glycaters [115,116]. Hemke [116] correctly points out the implication of these different phenotypes in the interpretation of an individual's HbA1c value, particularly when there are set targets for glycemic control, for example the ADA guidelines, which may not be relevant for all individuals. Smoking Syrjälä [117] conclude in their study that the combination of poor glycemic control, as seen as an increase in HbA1c, and smoking produce an increase in attachment loss of teeth in individuals with Type 1 diabetes [117]. In a large study conducted in Sweden, Nilsson et al. found that smoking was associated with both increased HbA1c values and microalbinuria, independent of other study parameters [118]. Iron deficiency Iron deficiency increases the HbA1c level [119], probably due to conservation of the red cells in iron deficiency, leading to increased red cell survival. Effects of drugs In HIV infection, glycemic control is underestimated in these patients [120] and is related to NRTI use. It had been reported that HbA1c is reduced in patients receiving ribavirin and peginterferonalpha-2 with hepatitis C [121,122]. An interesting case study shows a decrease in HbA1c level to 3.6% following commencement of therapy with ribavirin, peginterferonalpha-2a and erythropoietin in a previously well controlled patient with diabetes with HbA1c levels consistently between 6.0 and 7.0% and 3.6% [123]. The use of epoetin alpha and darbepoetin has been reported to decrease the HbA1c in an individual not undergoing hemodialysis [124]. Vitamin D levels are inversely associated with HbA1c levels according to two studies [125,126]. Since Vitamin D levels are lower in the winter, this coincides with the findings of lower HbA1c in the winter. Vitamin C has been reported to lower HbA1c levels [127,128] due to potentially inhibiting the glycation of hemoglobin. In some assays vitamin C may produce interference, falsely increasing HbA1c values [129]. Vitamin E may also inhibit glycation leading to lower HbA1c results [130]. Future directions and predictions for HbA1c On the analytical side, the imprecision of HbA1c methods will fall below 2% and may approach 1%. The throughput of analyzers performing HbA1c assay will increase and technologist involvement will decrease either with the use of middleware or algorithms built into the analyzer. The analytical performance of Point-of-Care instrumentation will improve to meet the demands of the marketplace.

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From a clinical perspective the amount of HbA1c testing performed for monitoring of glycemic control will increase due to the increasing number of individuals classified as diabetic. The use of HbA1c as a screening /diagnostic test will increase as the need for a test with minimal pre-analytical requirements is required for population screening for diabetes. As the precision and use of HbA1c expands, new interferences will be found and the number of factors that affect the analysis and interpretation such as ethnic difference will be found. The use of different reporting systems (SI and NGSP) will continue and the addition of estimated Average blood glucose (eAG) to the HbA1c report will not find wide acceptance [131]. References [1] Huisman TH, Martis EA, Dozy A. Chromatography of hemoglobin types on carboxymethylcellulose. J Lab Clin Med 1958;52:312–27. [2] Allen DW, Schroeder WA, Balog J. Observations on the chromatographic heterogeneity of normal adult and fetal hemoglobin: a study of the effects of crystallation and chromatography on the heterogeneity and isoleucine content. J Am Chem Soc 1958;80:1628–34. [3] Arnqvist H, Cederblad G, Hermanss G, Wettre S. Rapid and slow rate of decrease in HbA1a+b and HbA1c during improved glycaemic control. Scand J Clin Lab Invest 1982;42:265–71. [4] Brookchin RM, Gallop PM. Structure of hemoglobin A1c: nature of the N-terminal beta chain blocking group. Biochem Biophys Res Commun 1968;32:86–93. [5] Rahbar S, Blumenfeld O, Ranney HM. Studies of an unusual hemoglobin in patients with diabetes mellitus. Biochem Biophys Res Commun 1969;36:838–43. [6] Bunn HF, Haney DN, Gabbay KH, Gallop PM. Further identification of the nature and linkage of the carbohydrate in hemoglobin A1c. Biochem Biophys Res Commun 1975;67:103–9. [7] Koenig RJ, Peterson CM, Jones RL, Saudek C, Lehrman M, Cerami A. Correlation of glucose regulation and hemoglobin AIc in diabetes mellitus. N Engl J Med 1976;295:117–20. [8] Sikaris K. The correlation of hemoglobin A1c to blood glucose. J Diabetes Sci Technol 2009;3:429–38. [9] Diabetes Research Network Study Group. Relationship of A1c to glucose concentrations in children with Type 1 diabetes. Diabetes Care 2008;31:381–5. [10] Kilpatrick ES, Rigby AS, Atkin SL. Variability in the relationship between mean plasma glucose and HbA1c: implications for the assessment of glycemic control. Clin Chem 2007;53:697–901. [11] Tahara Y, Shima K. kinetics of HbA1c, glycated albumin, and fructosamine and analysis of their weight functions against preceding plasma glucose level. Diabetes Care 1995;18:440–7. [12] Goldstein DE, Little RR, Wiedmeyer H, England JD, Rohlfling C. Glycated haemoglobin estimation in the 1990s: a review of assay methods and clinical interpretation. In Marshall SM, Home PD, eds. The Diabetes Annual/8. Amsterdam: Elsevier Science B.V:193–212.1994. [13] Nording G, Dybkaer R. Recommendations for term and measurement unit of “HbA1c”. Clin Chem Lab Med 2007;45:1081–2. [14] Feiske A, Kobold U, Hoelzel W. Preparation of a candidate primary reference material for the international standardization of HbA1c. Clin Chem Lab Med 1998;36:299–308. [15] Feiske A, Kobold U, Hoelzel W. Preparation of candidate reference methods for hemoglobin A1c based on peptide mapping. Clin Chem 1997;43:1944–51. [16] Jeppsson JO, Kobold U, Barr J, Finke A, Hoelzel W, Hoshino T, et al. Approved IFCC reference system for measurement of haemoglobin A1c in human blood. Clin Chem Lab Med 2002;40:78–89. [17] Hoelzel W, Weykamp C, Jeppsson JO, Miedma K, Barr JR, Goodall I, et al. IFCC reference system for measurement of haemoglobin A1c in human blood and the national standardization schemes in the United States, Japan, and Sweden. Clin Chem 2004;50:166–74. [18] The American Diabetes Association, European Association for the Study of Diabetes, International Federation of Clinical Chemistry and Laboratory Medicine, the International Diabetes foundation. Consensus statement on the world wide standardization of the hemoglobin A1c measurement. Diabetes Care 2007;30:2399–400. [19] Sacks DB. Translating hemoglobin A1c into average blood glucose; implications for clinical chemistry. Clin Chem 2008;54:1756–8. [20] Barth JH, Der RI, Saudek CD. A randomized comparison of the terms estimated average glucose versus hemoglobin A1C. Diabetes Educ 2009;35:596–602. [21] Nathan DM, Kunen J, Borg R, Zheng H, Schoenfield D, Heine RJ. Translating the A1C assay into estimated average glucose values. Diabetes Care 2008;31:1–6. [22] Sacks DB, Bergenstal RM, McLaughlin S. The reporting of glucose with hemoglobin A1c. Clin Chem 2010;56:545–6. [23] Young IS. The reporting of estimated glucose with hemoglobin A1c. Clin Chem 2010;54:547–9. [24] Kilpatrick ES. Estimated average glucose (eAG) fit for purpose? Diabetes Med 2008;25:899–901. [25] Canadian Society of Clinical Chemists Position statement. Hemoglobin A1c Measurements and Reporting; March 2010.

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