GLYCATED HEMOGLOBIN (A1c/HbA1c/DOF-Hb) Methodologies, clinical reporting & analytical issues 7/27/2019 Dr Sikanaar Hayat Khan

Learning outcomes To improve upon the clinical usage of HbA1c What are the different methodologies for HbA1c? What should be out the analytical goals & standardization practices? What interpretation issues we can face in labs? How result should be reported & what treatment goals we should highlight as “chemical pathologists”? 7/27/2019 Dr Sikandar Hayat Khan

Formation Hb glycation occurs when glucose attaches to one or both of the N-terminal valines of the b-chain to form a SCHIFF’S BASE The process is fast and unstable and proceeds to form an IRREVERSIBLE KETOAMINE through Amadori rearrangement After a period of weeks, ADVANCED GLYCATION ENDPRODUCTS are formed and broken down 7/27/2019 Dr Sikandar Hayat Khan

CLINICAL USE Dr Sikandar Hayat Khan 7/27/2019

Points to ponder???? Can HbA1c be used for screening diabetes mellitus in all individuals? Can methodologies affect clinical decision making? What cut-offs are recommended for diagnosing diabetes mellitus? What cut-offs be recommending for monitoring diabetes mellitus? 7/27/2019 Dr Sikandar Hayat Khan

Clinical use Diagnosis of diabetes mellitus Monitoring of diabetes therapy SPECIAL SITUATIONS PREGNANCY ICU 7/27/2019 Dr Sikandar Hayat Khan

Why glycated hemoglobin??? 7/27/2019 Dr Sikandar Hayat Khan

Diagnosing diabetes International Expert Committee Report, drawn from the International Diabetes Federation (IDF) European Association for the Study of diabetes (EASD) American Diabetes Association (ADA), suggests the A1C level OF 48 mmol/mol (6.5 DCCT %) AS A DIAGNOSTIC LEVEL (The Committee Report further states that, when A1C testing cannot be done, the fasting and glucose tolerance tests be done) 7/27/2019 Dr Sikandar Hayat Khan

Diagnosing diabetes 7/27/2019 Dr Sikandar Hayat Khan

Diagnosing diabetes 7/27/2019 Dr Sikandar Hayat Khan

Points to ponder ??? What is the cost comparison of fasting blood glucose to glycated hemoglobin? Can POCT devices for A1c be used for diagnosis? Which parameter has more inter-lab variation? Which parameter has more biological variation? 7/27/2019 Dr Sikandar Hayat Khan

Points to ponder??? Dr Sikandar Hayat Khan

Individuals with stable glucose & A1C treatment goals Monitoring diabetes STABLE DIABETES UNSTABLE DIABETES Individuals with stable glucose & A1C treatment goals Individuals with unstable glucose & A1C treatment goals, like: Change of treatments Not meeting glycemic goals TWICE A YEAR QUARTERLY Dr Sikandar Hayat Khan

Monitoring diabetes therapy Personalized 7/27/2019 Dr Sikandar Hayat Khan

SPECIAL SITUATIONS DIAGNOSIS OF DIABETES DURING PREGNANCY continues to require fasting and glucose tolerance measurements for gestational diabetes, and not the glycated hemoglobin Hughes et al. Diabetes Care. 2014 Nov;37(11):2953-9. doi: 10.2337/dc14-1312. Dr Sikandar Hayat Khan

Hemoglobin types How diabetic monitoring be carried out in subjects known to have hemoglobinopathies with no/minimal amount of HbA? Different cut-offs? Different methodologies? Reliance of different parameter like glucose/fructosamine? Correct answer: Reliance of different parameter like glucose/fructosamine? In these patients a shortened red blood cell lifespan is typically encountered, which leads to a falsely low hemoglobin A1c 7/27/2019 Dr Sikandar Hayat Khan

Methodologies Dr Sikandar Hayat Khan 7/27/2019

Methods for glycated hemoglobin CHARGE BASED CATION-EXCHANGE RESIN CHROMATOGRAPHY ELECTROPHORESIS CATION-EXCHANGE HPLC STRUCTURE BASED BORONATE AFFINITY CHROMATOGRAPHY AFFINITY ELECTROPHORESIS IMMUNOASSAYS CHEMICAL METHODS REFERENCE METHOD IFCC TANDEM MASS SPECTROMETRIC ASSAY POCT IMMUNOASSAY 7/27/2019 Dr Sikandar Hayat Khan

Methods for glycated hemoglobin CHARGE BASED STRUCTURE BASED Cation-exchange resin chromatography Electrophoresis Cat ion-exchange HPLC Boronate affinity chromatography Affinity electrophoresis Immunoassays 7/27/2019 Dr Sikandar Hayat Khan

Methods for glycated hemoglobin CHARGE BASED STRUCTURE BASED More susceptible to interference from Hb variants More susceptible to post-translational modifications (e.g., carbamylation and acetylation) Boronate affinity chromatography Less susceptible to interference from Hb variants Immunoassay Antibodies target the B-N terminal of first 4-10 AA of the glycated hemoglobin Caution with regards to certain ethnicities 7/27/2019 Dr Sikanadar Hayat Khan

POCT devices Nycocard i-chroma DCA Vantage In2 It (Biorad) A1cNow (Bayer) 7/27/2019 Dr Sikandar Hayat Khan

POCT devices 7/27/2019 Dr Sikandar Hayat Khan

ANALYTICAL GOALS & STANDARDIZATION Dr Sikandar Hayat Khan 7/27/2019

Points to ponder??? Do we face inter-lab variation in our A1c reporting? What specific reason do we have for inter-lab variation? Do we think of calibration issues while installing a new kit program in our labs? Does it matter? What IFCC has to say about calibration? What is primary reference method for HbA1c ? 7/27/2019 Dr Sikandar Hayat Khan

An assay standardization issue When the test was brought in-house and results reported began to be slightly higher than they had been, a trend appeared in which the overall results drifted down (ie, patients intensified their efforts). The opposite effect was observed when the calibration was changed and results appeared lower than they had been previously. Then a trend appeared in which the overall results drifted up (ie, patients allowed their glycemic control to deteriorate). Clearly patients (and physicians) respond to changes in hemoglobin A1c results. Multiple changes in treatment and associated psychological changes were faced by patients due to A1c methodology& calibration changes Hanas R et al. Diabetes Care. 2002;25:2110-2111. 7/27/2019 Dr Sikandar Hayat Khan

Precision targets Program must be able to achieve a CV below 3% at HbA1c levels of 6% and 9% Manufacturers’ assays should be able to achieve a CV below 5% Reference: IFCC AND AACC International Standardization Committee Dr Sikandar Hayat Khan

Comparison of precision Check your kits and methods? Everybody is different? 7/27/2019 Dr Sikandar Hayat Khan

A1c Inter-lab variation CVs of 5%–7% has been shown for HbA1c values between 6% and 10% Poor between-lab agreement can also be found for the same manufacturer’s method IFCC established a Working Group (IFCC WG-HbA1c) to achieve international standardization of HbA1c measurement Mosca A at al. Global standardization of glycated hemoglobin measurement: the position of the IFCC Working Group. Clin Chem Lab Med.2007;45(8):1077-80. 7/27/2019 Dr Sikandar Hayat Khan

IFCC standardization program (HbA1c) Achievements of this WG includes: Development of highly purified primary Reference Materials (HbA1c and HbA0) by Institute for Reference Materials and Measurements (IRMM) A reference measurement procedure for HbA1c has been developed as: - Proteolytic digestion of red cell hemoglobins FOLLOWED BY -Quantitative peptide mapping by: HPLC-mass spectrometry OR HPLC-capillary electrophoresis It has been voted on by the National Societies affiliated to the IFCC and published as an ‘‘APPROVED IFCC REFERENCE MEASUREMENT PROCEDURE’’ 7/27/2019 Dr Sikandar Hayat Khan

IFCC standardization program (HbA1c) Achievements of this WG includes: Development of highly purified primary Reference Materials (HbA1c and HbA0) by Institute for Reference Materials and Measurements (IRMM) A reference measurement procedure for HbA1c has been developed as: - Proteolytic digestion of red cell hemoglobins FOLLOWED BY -Quantitative peptide mapping by: HPLC-mass spectrometry OR HPLC-capillary electrophoresis It has been voted on by the National Societies affiliated to the IFCC and published as an ‘‘APPROVED IFCC REFERENCE MEASUREMENT PROCEDURE’’ 7/27/2019 Dr Sikandar Hayat Khan

IFCC standardization program (HbA1c) c) Provision to IFCC lab network for further development of secondary reference materials Two experiments are performed every year in which materials are distributed to the laboratories for: Comparison purposes To assign HbA1c values to candidate calibrators and controls The network has developed: SET OF RULES for the certification of reference values To obviate uncertainties in CALCULATION OF CALIBRATORS Dr Dikandar Hayat Khan

IFCC standardization program (HbA1c) d) Comparison studies have been performed between the IFCC reference measurement laboratories and the existing Designated Reference Method (DCMs) These studies found stable relationships between the IFCC and different (DCM) systems and the corresponding regression equations (the ‘‘master equations’’) were published Dr Dikandar Hayat Khan

IFCC standardization program (HbA1c) e) Secondary Reference Materials (SRMs) have been produced in the form of panels of fresh and frozen whole blood and distributed to the manufacturers and to laboratories performing DCMs to anchor their methods to the IFCC reference system Dr Dikandar Hayat Khan

IFCC standardization program (HbA1c) IRMM IFCC lab netwrok IFCC reference measurement system and traceability chain for HbA1c Dr Dikandar Hayat Khan

Name and units for the IFCC standardized HbA1c test Recently, a recommendation by the IFCC-IUPAC Committee on Nomenclature, Properties and Units (C-NPU) has been prepared that relates to the: SYSTEMATIC UNITS FOR HbA1c(as measured by IFCC ref procedure) 7/27/2019 Dr Sikandar Hayat Khan

Name and units for the IFCC standardized HbA1c test (NOMENCLATURE) What compound do we have in mind once we say glycated hemoglobin? Fraction of the b-chains of hemoglobin that has a stable hexose adduct on the N-terminal amino acid valine Thus it can be expressed as ‘‘Hemoglobin beta chain(Blood)—N-(1-deoxyfructos-1-yl) ’’ i.e., bN1-deoxyfructosyl-hemoglobin (DOF-Hb) Reference: IFCC-IUPAC (C-NPU) document on glycated hemoglobin The IFCC WG-HbA1c agrees with the use of this nomenclature because it gives tracebility to the glycated hemoglobin to original IFCC reference procedure 7/27/2019 Dr Sikandar Hayat Khan

Name and units for the IFCC standardized HbA1c test (UNITS) IFCC supports the use of ‘‘millimole per mole’’ will be chosen instead of ‘‘percent’’ (%) According to IFCC to avoid confusion ? Converting to SI units Broad range for explanation of results 7/27/2019 Dr Sikandar Hayat Khan

RESULT REPORTING & treatment goals Dr Sikandar Hayat Khan 7/27/2019

Reporting HbA1c as eAG DCCT trial and UKPDS data indicated a direct relationship between HbA1c and Mean Blood Glucose 1% change of HbA1c = 25 -35% change in average plasma glucose* MBG = 33.3 (HbA1c) - 86 (r=0.958) ** Major advantage by reporting HbA1c as an eAG is that both physicians and patients understand glucose *(Goldstein et al, Clin Chem 1986;32:B64) **(Nathan et al, NEJM 1984;310;341) Dr Dikandar Hayat Khan

Translating HbA1c as eAG The A1c Assay into estimated average glucose values* A1c determination in the A1c Derived Average Glucose (ADAG)** *Diabetes Care 2008; 31:`473-1478 **Clin Chem Lab Me 2008’46:1617-1623 Dr Dikandar Hayat Khan

eAG limitations Ethnic data (Most studies on Caucasians) No data from Children Pregnancy Renal failure Concept of “glycation status” High glycators vs low glycators MBG Mean HbA1c 95% CI HbA1c 154 mg/dl 7.0% 6.7-9.2% high glycator will have a much higher A1c than the low glycator and vice versa * *Hempe JM et al. J Diabetes Complications 2002;16(5):313-20. Dr Dikandar Hayat Khan

Measuring eAG eAG (mmol/l) = 1.59 × A1C − 2.59 Dr Dikandar Hayat Khan

eAG (estimated average glucose) Measuring eAG HbA1c eAG (estimated average glucose) (%) (mmol/mol) (mmol/L) (mg/dL) 5 31 5.4 (4.2–6.7) 97 (76–120) 6 42 7.0 (5.5–8.5) 126 (100–152) 7 53 8.6 (6.8–10.3) 154 (123–185) 8 64 10.2 (8.1–12.1) 183 (147–217) 9 75 11.8 (9.4–13.9) 212 (170–249) 10 86 13.4 (10.7–15.7) 240 (193–282) 11 97 14.9 (12.0–17.5) 269 (217–314) 12 108 16.5 (13.3–19.3) 298 (240–347) 13 119 18.1 (15–21) 326 (260–380) 14 130 19.7 (16–23) 355 (290–410) 15 140 21.3 (17–25) 384 (310–440) eAG Endorsements American Diabetes Association (ADA) International Diabetes Federation (IDF) American Association of Clinical Chemistry (AACC) International Federation of Clinical Chemistry (IFCC) European Association for the study of Diabetes (EASD) 7/27/2019 Dr Sikandar Hayat Khan

IFCC vs DCCT confusion 7/27/2019 Dr Sikandar Hayat Khan

IFCC vs DCCT confusion Switch to IFCC units HbA1c is to be reported in the IFCC units* IFCC reporting was introduced in Europe except for the UK in 2003 UK carried out dual reporting from 1st June 2009 until 1 October 2011 (Still going on in most set ups) *Guidelines by ADA, EASD, IFCC and IDF 7/27/2019 Dr Sikandar Hayat Khan

IFCC vs DCCT confusion So if u have a DCCT certified kit reporting a HbA1c result of 9% (75 mmol/mol) then what will be his IFCC A1c values = ???? (6.85%) 74.86 mmol/mol The 2010 ADA Standards of Medical Care in Diabetes added the A1c ≥ 48 mmol/mol (≥6.5 DCCT %) as another criterion for the diagnosis of diabetes Dr Dikandar Hayat Khan

A reporting pattern for HbA1c Dr Dikandar Hayat Khan

Reporting data for A1C The data to be provided with HbA1c must include following: Results as per DCCT (%) Results as per IFCC (mmol/mol) Methodology type HbA1c types included eAG Appropriate references Diagnostic & therapeutic cut-offs Interpretation & opinion 7/27/2019 Dr Sikandar Hayat Khan

Diagnostic & therapeutic goals Dr Dikandar Hayat Khan

Interpretation issues Dr Sikandar Hayat Khan 7/27/2019

Consideration Your method type? Clinical need? Patient type? Charge based? Non-charge based? POCT ? Clinical need? Diagnosis? Monitoring? Patient type? Type-1 diabetic Type-2 diabetic Pregnancy? Underlying ailments? Red cell disorder (Hemoglobinopathies?) Uremia? IDA? 7/27/2019 Dr Sikandar Hayat Khan

Concept of interpretation A1c RBC related Glucose bonding Analytical targets 7/27/2019 Dr Sikandar Hayat Khan

Patient condition affecting A1c results Increase Decrease Splenectomy *# Thalassemia *# HbF*# IDA* Uremia (Carbamylated Hb)* Alcoholism* Hyperbilirubinemia* Hypertriglyceridemia* Lead poisoning* Large dose of salicylates (Acetylated Hb)*# Opiate addiction* Hemolytic anemia Acute blood loss HbC* HbS* Pregnancy *ion-exchange #electrophoretic method 7/27/2019 Dr Dikandar Hayat Khan

Medications affecting A1c results Glycation Sulfation Acetylation Carbamylation 7/27/2019 Dr Sikandar Hayat Khan

Influences on “Glycation Process” RBC’s half-life Hemolysis Hemoglobinopathies Anemia B12/Folate deficiency Blood glucose Glucose permeability (Door to glucose entry into cell) Hb G Permeability of the RBC to glucose influences the amount of glycation and could explain the discordance noted in some hematologically normal people with diabetes 7/27/2019 Dr Sikandar Hayat Khan

Assessment Question Your Reply Any method of A1c reporting can be used for diagnosis of diabetes mellitus (YES/NO) DOF-Hb has been endorsed by IFCC for use IFCC (YES/NO) What A1c cut-off has been recommended by DCCT for diabetes diagnosis? SI units for reporting A1c are __________ Glycation rates are not different among various ehnicities (TRUE/FALSE) 7/27/2019 Dr Sikandar Hayat Khan

Assessment Question Your Reply A1c reporting needs to be standardized to improve accuracy (YES/NO) Drugs causing hemolysis can decrease Aic results (YES/NO) Precision recommended by IFCC as a n anlytical target for A1c should be? A patient with long standing iron deficiency anemia can have lower A1c results? (TRUE/FALSE) What kind of bias the IFCC result will have in comparison to DCCT-certified results? 7/27/2019 Dr Sikandar Hayat Khan

Assessment Question Your Reply Hypertriglyceridemia and hyperbilirubinemia can increase A1c results? (YES/NO) IFCC reference method for A1c is ______ Boronate affinity chromatography is a charge based method. (TRUE/FALSE) Stable diabetes is monitored by twice yearly testing for A1c (TRUE/FALSE) Precision issues in case of A1c are more pronounced at higher values (TRUE/FALSE) 7/27/2019 Dr Sikandar Hayat Khan

Thanks a lot 7/27/2019 Dr Sikandar Hayat Khan