Biochemical tests in diabetes HBA 1 c standardization

Biochemical tests in diabetes HBA 1 c standardization Dr Javad Mohiti Dept of Clinical Biochemistry

Standardization of HBA 1 c 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)

Glycated Proteins Glycated Hemoglobin Hb. A(97. 5%) Hb. A 2(2. 5%) Hb. F(0. 5%) Hb. A(Hb. A 1 a, Hb. A 1 b, Hb. A 1 c=Hb. A 1) Hb. A 1 c 80%

Pre-Hb. A 1 c(3 -5%Normal, 8 -30% Diabetes) Clinical use of Hb. A 1 c §

Methods of Measurement of Hb. A 1 c 1. 2. 3. Different charge(Chromatogeraphy, Electr. ) Different structure (immunoassay) Chemical Ion exchange che. -Cat. ion exch. -anion exchange (Pre-Hb. A 1 c, Hb. F with Hb. A 1) Alchol, Pb, Aspirin)

Ion-exchange chromatography Measures HBA 1 – total glycated haemoglobins (A 1 a + 1 b + 1 c) HPLC Both Hb. A 1 c and Hb. A 1 can be reported, Mass spectroscopy Capillary Elec

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 Hb. A 1 c

Diabetes Control and Complications Trial (DCCT) 19950 -2010 multicenter randomized trial Hb. A 1 c 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 Hb. A 1 c The method should be a DCCT –aligned HBA 1 c method The assay should have acceptable within assay precision <3% and between assay imprecision <5%

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 are 1. 5 -2. 0% lower than NGSP Clinical Chemistry 2008; 54: 240 Update 6 year progress report IFCC recommends mmol/mol Hb. A 1 c as units

Interference Icterus : Lipemia Hemoglobin variants S and C have no effect on the assay when they exist in the heterozygous forms Hb. AS and Hb. AC. In homozygous Hb SS or Hb CC patients do not have Hb. A present or Hb. A 1 c thus criteria other than monitoring of Hb. A 1 c must be used to assess long term diabetic control in these patients. Hb. F levels upto 30 % do not interfere

A 1 c (%) Mean Plasma Glucose mg/dlmmol/l 61267. 5 71549. 5 818311. 5 921213. 5 1028515. 5 1132017. 5 1235519. 5

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 23 weeks Do not perfom when Albumin < 3 g/d. L

WBG 12 -15% less than plasma glucose. Loss of glucose approx 5 -7% per hour (5 -10 mg/d. L) 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 40 C or 24 hours at 15 -250 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

Reference Values ADA 2 fasting plasma values ≥ 126 mg/d. L (7. 0 mmol/L) Impaired fasting glucose mmol/L) 101 - 124 mg/d. L (5. 6 -6. 9 Glucose AC fasting 70 -110 mg/d. L Glucose PC (2 hours) 80 -140 mg/d. L Glucose random 70 -140 mg/d. L

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 234 nm Specimen type, collection and storage Random urine sample. Stability one week at 40 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.

g/min mg/24 hr <20 <30 20 -200 30 – 300 >200 >300 mg/g <30 normal 30 – 300 increased UAE >300 overt diabetic nephropathy

MEASUREMENT OF Sampleling (Whole blood, 1012%↓, vessle, Glycolysis 5 -7%, Iodoacetate GLUCOSE 1 -Hexokinse 2 -Glucose Oxidase 3 -Glucose dehydrogenase Self monitoring blood Glucose Measurement of glucose in urine