1. Biochemical tests in diabetes HBA1c standardization Dr Javad Mohiti Dept of Clinical Biochemistry

2.  Availability of glycated heamoglobin measurment has been one of the major advance  diabetes in last 30 years  The diabetes control and complication Trial(DCCT)  International Federation of clinical  chemistry(IFCC)  National Glycohemoglobin Standarization Program(NGSP)

3.  Glycated Hemoglobin  HbA(97.5%)  HbA2(2.5%)  HbF(0.5%)  HbA(HbA 1a,HbA 1 b,HbA1c=HbA 1 )  HbA1c 80%

5.  Pre-HbA1c(3-5%Normal, 8-30% Diabetes)  Clinical use of HbA1c

6. 1. Different charge(Chromatogeraphy, Electr.) 2. Different structure (immunoassay) 3. Chemical  Ion exchange che.  -Cat. ion exch.  -anion exchange  (Pre-HbA1c, HbF with HbA1)  Alchol,Pb,Aspirin)

7. Ion-exchange chromatography Measures HBA1 – total glycated haemoglobins (A1a + 1b + 1c) HPLC Both HbA1c and HbA1 can be reported, Mass spectroscopy Capillary Elec

8. Immunoassay antibodies raised against the Amadori product of glucose (ketoamine linkage) plus the first 4-8 amino acids at the N-terminal of the beta chain by inhibition of latex agglutination. Specific for HbA1c

9. Diabetes Control and Complications Trial (DCCT) 19950-2010 multicenter randomized trial HbA1c measurement systems have been standardized through a process of alignment with the original DCCT method. This has been undertaken by the US National Glycohemoglobin Standardisation Program (NGSP). UK Consensus Statement Glycemic control is best measured by HbA1c The method should be a DCCT –aligned HBA1c method The assay should have acceptable within assay precision <3% and between assay imprecision <5%

10. NGSP = 0.0906(IFCC) + 2.21. This method reports performance data and reference ranges as NGSP values. The calibrator/diluent set includes both NGSP and IFCC values. IFCC values are 1.5 -2.0% lower than NGSP Clinical Chemistry 2008; 54:240 Update 6 year progress report IFCC recommends mmol/mol HbA1c as units

11. Interference Icterus : Lipemia Hemoglobin variants S and C have no effect on the assay when they exist in the heterozygous forms HbAS and HbAC. In homozygous Hb SS or Hb CC patients do not have HbA present or HbA1c thus criteria other than monitoring of HbA1c must be used to assess long term diabetic control in these patients. HbF levels upto 30 % do not interfere

12.  mg/dlmmol/l  61267.5  71549.5  818311.5  921213.5  1028515.5  1132017.5  1235519.5

13. Fructosamine Generic name for plasma protein ketoamines Glucose and ε lysine residues of albumin Half life of circulating albumin is 20 days Glycated albumin reflects control over a period of 2- 3 weeks Do not perfom when Albumin < 3 g/dL

14. WBG 12-15% less than plasma glucose. Loss of glucose approx 5-7% per hour (5-10 mg/dL) Fasting blood glucose (FBG) should be 10 hour fast not 16 hrs EDTA/Fluoride specimen is stable for 7 days is a closed tube at 4 0 C or 24 hours at 15-25 0 C. CSF should be analysed within 2 hours. Hexokinase and GOD/POD methods are not suitable for urine. Clin Chem 2005; 51:1573-1576 Harmonisation of POCT devices with laboratory use a factor of 1.11 to convert POCT values in whole blood to plasma values

15. Reference Values ADA 2 fasting plasma values ≥ 126 mg/dL (7.0 mmol/L) Impaired fasting glucose 101- 124 mg/dL (5.6-6.9 mmol/L) Glucose AC fasting70-110 mg/dL Glucose PC (2 hours)80-140 mg/dL Glucose random 70-140mg/dL

16. Estimation of urine microalbumin Summary and explanation of the test Immunoturbimetric assay. In solution the precipitate formed by an antigen-antibody complex between albumin in the urine and albumin antibody scatters light. The intensity of transmitted light is compared to that of the incident light. The antigen antibody reaction is enhanced by polyethylene glycol Absorbance is measured at 234nm Specimen type, collection and storage Random urine sample. Stability one week at 4 0 C. Source of the Method Protocol Based on the optimised standard method of Van Munster PJJ et al Clin Chim Acta 76,377-388, 1977.

17.  g/minmg/24hrmg/g <20<30<30 normal 20-20030 – 30030 – 300 increased UAE >200>300>300 overt diabetic nephropathy

18. Sampleling (Whole blood, 10- 12%↓,vessle,Glycolysis5-7%,Iodoacetate 1-Hexokinse 2-Glucose Oxidase 3-Glucose dehydrogenase Self monitoring blood Glucose Measurement of glucose in urine