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

Biochemical tests in diabetes

HBA1c standardizationDr Javad Mohiti

Dept of Clinical Biochemistry

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 HemoglobinHbA(97.5%)HbA2(2.5%)

HbF(0.5%)HbA(HbA1a,HbA1b,HbA1c=HbA1)

HbA1c 80%

Pre-HbA1c(3-5%Normal, 8-30% Diabetes)Pre-HbA1c(3-5%Normal, 8-30% Diabetes)Clinical use of HbA1cClinical use of HbA1c

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)

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

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 StatementGlycemic control is best measured by HbA1cThe method should be a DCCT –aligned HBA1c methodThe 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.

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

InterferenceIcterus : 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

mg/dlmmol/l61267.571549.5818311.5921213.51028515.51132017.51235519.5

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 2-3 weeks

Do not perfom when Albumin < 3 g/dL

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 hrsEDTA/Fluoride specimen is stable for 7 days is a closed tube at 40C or 24 hours at 15-250C.CSF should be analysed within 2 hours. Hexokinase and GOD/POD methods are not suitable for urine.

Clin Chem 2005; 51:1573-1576Harmonisation 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/dL (7.0 mmol/L)

Impaired fasting glucose 101- 124 mg/dL (5.6-6.9 mmol/L)

Glucose AC fasting 70-110 mg/dL

Glucose PC (2 hours) 80-140 mg/dL

Glucose random 70-140mg/dL

Estimation of urine microalbumin

Summary and explanation of the testImmunoturbimetric 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 storageRandom urine sample. Stability one week at 40C.Source of the Method ProtocolBased on the optimised standard method of Van Munster PJJ et alClin Chim Acta 76,377-388, 1977.

g/min mg/24hr mg/g

<20 <30 <30 normal

20-200 30 – 300 30 – 300 increased UAE

>200 >300 >300

overt diabetic nephropathy

Sampleling (Whole blood, 10-12%↓,vessle,Glycolysis5-7%,Iodoacetate

1-Hexokinse2-Glucose Oxidase

3-Glucose dehydrogenaseSelf monitoring blood GlucoseMeasurement of glucose in urine