HbA1c and Diabetes. Steven Weier Senior Lecturer School of Biomedical Sciences Faculty of Health QUT. CRICOS No J

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1 HbA1c and Diabetes Steven Weier Senior Lecturer School of Biomedical Sciences Faculty of Health QUT Queensland University of Technology

2 History of HbA1c 1966: Holmquist and Schroeder identify five subtypes of haemoglobin A, including HbA1c. 1968: ahbar recognises that HbA1c is elevated in people with diabetes. 1975: Koenig and Cerami suggest that HbA1c is related to metabolic control : HbA1c assays become commercially available 1993: DCCT establishes HbA1c as a valuable clinical marker in people with type 1 diabetes. 1998: UKPDS establishes HbA1c as a valuable clinical marker in people with type 2 diabetes.

3 What is HbA1c? Glucose attaches to N-terminal valine residue of one or both beta chains of haemoglobin via a nonenzymatic, post-translational process: A reversible reaction leads to the formation of an aldimine (Schiff Base) Followed by an Amadori rearrangement to form an irreversible ketoamine. The resultant product is called HbA1c HbA1c concentration depends on: Life span of BC Glucose concentration

4 HbA1c

5 Normal Haemoglobin Fractions Haemoglobin HbA (alpha2,beta2): 95-98% HbA2 (alpha2,delta2): % HbF (alpha2,gamma2): <2% There are several minor haemoglobins migrate more rapidly than HbA in an electric field called HbA1. HbA1 is made up of HbA1a + HbA1b + HbA1c.

6 HbA1 vs. HbA1c HbA1a1 is fructose-1,6 diphosphate attached to the N-terminal of the beta chain. HbA1a2 is glucose-6-phosphate attached to the N- terminal of the beta chain. HbA1b contains adducts of pyruvic acid at the amino terminus of the α chain HbA1c is glucose attached to the N-terminal valine of each beta chain of haemoglobin HbA1c makes up >90% of HbA1. Normally less than 6% of Hb is HbA1c

7 HbA1c and Diabetes HbA1c levels are elevated in diabetics Levels indicate how well a diabetic patient is managed (i.e. glycaemic control) Levels also correlate with the risk of developing complications associated with diabetes To the point it has evolved to become an essential tool for prognosis, monitoring, treatment, and diagnosis of diabetes mellitus.

8 Diabetes Complications etinopathy (Blindness) Nephropathy (CF) Peripheral neuropathy Feet ulceration and/or amputations Hypertension Hyperlipidaemia Cardiovascular disease

9 HbA1c & isk of Complications

10 HbA1c & isk of Complications

11 HbA1c Assays Immunoassay Cation Exchange Chromatography Affinity Chromatography Enzymatic Capillary Electrophoresis

12 Immunoassay Immunoturbimetric (Latex Agglutination Inhibition Assay) An agglutinator, consisting of a synthetic polymer containing multiple copies of the immunoreactive portion of HbA1c, causes agglutination of latex coated with HbA1c specific mouse monoclonal antibodies. In the absence of HbA1c in the sample, the antibody-coated microparticles and the agglutinator will agglutinate. Agglutination leads to an increase in the turbidity of the suspension. The presence of HbA1c in the sample results in a decrease in the rate of agglutination of the antibody-coated microparticles and the agglutinator. The increase in turbidity and therefore absorbance is, inversely proportional to the concentration of HbA1c in the sample. In most systems the antibody recognises the valine in the N-terminal position of the beta globin chain Measures HbA1c Maybe unsuitable for some patients with haemoglobin variants (method dependant).

13 Latex Agglutination Inhibition Assay

14 Cation Exchange Chromatography Ion Exchange (Cation Exchange): The adsorption of the molecules to the solid phase is driven by the ionic interaction between the oppositely charged ionic groups in the sample molecule and in the functional ligand on the support. Cation Exchange = Negatively charged stationary phase, positively charged analyte The strength of the interaction is determined by the number and location of the charges on the molecule and on the functional group. By increasing the ionic strength of the mobile phase (generally by using a linear gradient) the molecules with the weakest ionic interactions start to elute from the column first. Molecules that have a stronger ionic interaction require a higher salt concentration and elute later in the gradient. Most systems still have issues with variants but analysis allows for simultaneous identification of these patients. Current generation systems do not suffer from interference from carbamylated haemoglobin or Labile A1c (Schiff Base) Both HbA1c and HbA1 can be reported.

15 Cation Exchange Chromatography

16 Affinity Chromatography Boronate Affinity In this analytical technique, a boronate such as phenylboronic acid is bonded to the surface of the column support. When the sample haemolysate is passed through the column, glycosylated HbA is retained by the complexing of its diol groups with the boronate After the unretained non-glycated component elutes from the column, the glycated component is eluted from the column with a reagent (e.g. sorbitol) that displaces it from the boronate. Measures HbA1 NOT HbA1c

17 Affinity Chromatography

18 Affinity Chromatography

19 Direct Enzymatic Method Whole blood sample is lysed and then subjected to extensive proteolytic digestion. This process releases amino acids, including glycated valines, from the haemoglobin beta chains. The glycated valines serve as substrates for a specific recombinant enzyme; fructosyl valine oxidase (FVO). FVO specifically cleaves N-terminal valines and produces hydrogen peroxide. The detection step is a peroxidase (POD) catalysed reaction with a suitable chromagen and is measured spectrophotometrically.

20 Capillary Electrophoresis Capillary electrophoresis uses the principle of liquid flow capillary electrophoresis in free solution. With this technique, molecules are separated on the basis of its quantity of charge compared to its relative hydrodynamic size. Hydrodynamic size very closely relates to the mass of the molecule CE has incredible efficiency and ability to separate similarly structured compounds. This is due to Electro-Osmotic Flow (EOF) When a voltage is applied across a tube filled with an electrolyte solution, the solution begins to move toward the cathode. For HbA1c, separation it is conducted in an alkaline buffer at a specific ph (~9.4). The migration takes place in silica capillary tubes with a small internal diameter ( 25μm). The migration generally takes places at a very high voltage (e.g volts) in a temperature controlled environment. The haemoglobins are detected and measured at 415 nm at the cathodic end of the capillary.

21 Capillary Electrophoresis

22 Capillary Electrophoresis

23 HbA1c Standardisation Following the introduction of HbA1c methods into routine use, it quickly become apparent that there was a significant difference in the results produced by different laboratories. It was evident that the disparate results obtained were due to the range of methods being used by laboratories and the lack of a primary reference material. Standardisation with common calibration was first proposed in 1984 by Peterson et al., who examined factors such as inter-method correlation and reproducibility between laboratories. However, it was only after the publication of the DCCT study in 1993 that the issue of international standardisation of glycated haemoglobin measurements became an important objective for scientists and clinicians. The lack of international standardisation resulted in several countries developing national standardisation programs.

24 Standardisation National Glycohaemoglobin Standardisation Program (NGSP) Formed in July 1996 to implement recommendations developed by the AACC Glycohemoglobin Standardisation Subcommittee. Established a network of reference laboratories that would calibrate and standardise commercial HbA1c methods and analytical systems. The Bioex 70 HPLC System, established in the DCCT Central Laboratory, was chosen as the NGSP reference standard method. The NGSP is responsible for the calibration of HbA1c methods in many parts of the enabling direct comparison to the DCCT targets. There were also similar national programs in Japan and Sweden.

25 Global Standardisation Standardisation These national programs had an absence of internationally recognised and accepted reference materials and measurement procedures to assure the accuracy and comparability of HbA1c measurements at a global level. IFCC established a Working Group on HbA1c Standardisation (WG- HbA1c) with the aim to develop a complete reference measurement system based on the concepts of metrological traceability. HbA1c was defined as haemoglobin molecules having a stable adduct of glucose to one or both of the N-terminal valine of the haemoglobin β chain (β N-1-deoxyfructosyl-haemoglobin). The rationale was that this quantity is biochemically well characterised, it is the major form of HbA1c in human blood, and most of the commercial HbA1c tests claim to measure only this form.

26 Global Standardisation Standardisation Two reference methods were developed which specifically measure the glycated N-terminal residue of the beta-chain. The principle is that in a first step hemoglobin is cleaved into peptides by a proteolytic enzyme and thereafter the specific glycated and nonglycated N-terminal peptides of the beta-chain are measured by HPLC and either mass spectrometry or capillary electrophoresis. The WG-HbA1c was also successful in preparing primary reference materials (purified HbA0 and HbA1c) to calibrate the reference procedures. In 2001, the IFCC reference methods were unanimously accepted by the National Societies of Clinical Chemistry following a ballot and published as approved IFCC reference methods.

27 Standardisation The relationships of each national designated comparison method (DCM) with the IFCC method have been stable and reproducible over several years, and are described by the following master equations

28 eporting Units The joint IFCC Committee on Nomenclature, Properties and Units and IUPAC (International Union of Pure and Applied Chemistry) Subcommittee on Nomenclature, Properties and Units (C-NPU) proposed mmol/mol be used as the unit of measurement for HbA1c; this represents the SI unit for this analyte. This option was chosen to avoid confusion when recalculating old HbA1c targets to the new IFCC standardised values if clinical laboratories wish to implement HbA1c results traceable to the IFCC reference system. The other advantage of this approach may include a positive impact of changing of scale of reported HbA1c results allowing clinicians and diabetic patients to better understand the analyte changes. Currently clinicians and patients may perceive HbA1c small changes as unimportant, although they are linked to large health effects.

29 eporting Units

30 eporting Units In 2011 a joint statement from the Australasian Association of Clinical Biochemists, the Australian Diabetes Educators Association, the Australian Diabetes Society and the oyal College of Pathologists of Australasia recommended that: From July 2011, HbA1c levels should be reported in both National Glycohemoglobin Standardization Program units (percentage) and the Système International (SI) units (mmol/mol) by all pathology laboratories and, where possible, from point-of-care devices. The period of dual reporting will be 2 years, after which only the SI units will be used. These recommendations were consistent with international recommendations and those already in place in New Zealand.

31 Target Values <6.0 % (<42) Non-diabetic <7.0 % (<53) Treatment Target in diabetics >8.0 % (>64) - Change of therapy in diabetics

32 Conditions that Preclude HbA1c Testing Altered red blood cell turnover e.g. haemolytic anaemia, major blood loss or blood transfusion Splenectomy Haemoglobin variants (depending on the method) enal Failure Liver Failure Pregnancy egular phlebotomy Haemochromatosis egular blood donor

33 HbA1c for Diagnosis In 2011 WHO recommended that HbA1c can be used as a diagnostic test for diabetes providing that: Stringent quality assurance tests are in place and Assays are standardised to the international reference values, and There are no conditions present which preclude its accurate measurement. WHO established the following cut-offs: Non-diabetic: < 6.1% (43 mmol/mol) Impaired glucose metabolism: % (43-46 mmol/mol) Diabetes mellitus: 6.5% (48 mmol/mol)

34 Caveats: HbA1c for Diagnosis Not recommended for general screening Not an appropriate test to confirm diagnosis of diabetes in patients with: Significant chronic medical conditions Any anaemia or any abnormality of red cell structure In these patient fasting, random glucose levels and OGTT would be required for diagnosis From 1 st of November 2014 HbA1c has now been recognised by Medicare as a diagnostic test As a diagnostic test, up to 1 test in a 12 month period is rebatable by Medicare. For monitoring established diabetes the limit of 4 tests per year remains unchanged.

35 Benefits of HbA1c vs Glucose for Diagnosis Low pre-analytical and biological variation CV I - 1.9% (Westgard QC icos Database) Values reflect chronic glycaemia No requirement for a special pre-test preparation such as diet or a fasting sample. HbA1c is stable when collected in the appropriate specimen tube. 1week at C

36 Thank You