1. INSTRUCTIONS TO PATIENT  Explain to the patient the purpose of test and the need for a blood sample to be drawn.  No fasting is required before the test.

2. PROCEDURE  A 3-5-ml blood sample is drawn through a vene puncture to a sodium floride oxalate tube.  Gloves are worn throughout the procedure  Apply pressure at Venopuncture site to arrest bleeding  Label the sample and transport to the Lab.

3. VALUE OF TEST  To determine the degree of glucose control.  Screening for DM.  Distinguishing chemical forms from overt Diabetes.  Determining the relationship between chronic high blood sugar level and pathological complications of DM.  Evaluating the efficiency of diabetic therapy.  Managing DM in Pregnancy.

4. PRINCIPLE OF GLYCATION  Glycation of protein is a frequent occurrence, but in the case of haemoglobin, a non- enzymatic seaction occurs betweeen glucose and N-end of the beta-chain. This form a Schiff base which is itself conserted to 1- deoxyfructose. This rearrangement is an example of an Amadori rearrangement.

5. CONTD.  When blood glucose levels are high, glucose molecules attach to the Hb in red blood cells. The longer hyperglycemia occurs in blood, the more glucose binds to Hb in the red blood cells and the higher the gycated haemoglobin.

6. CONTD.  Once a haemoglobin molecule is glycated, it remains that way. A build-up of HbA1c WITHIN the red cell, therefore, reflects the average level of glucose to which the cell has been exposed during its life-cycle. Measuring HbA1c assesses the effectiveness of therapy by monitoring long-term serum glucose regulation.

7. LIMITATION  Although HbA1c levels are reliable indication of recent average glycaemic control, it does not provide information about the daily pattern of blood glucose levels, Therefore information required for logical adjustment of insulin doses is derived from Routine RBS, FBS test

8. CONTD.  More recent changes in glycaemia (ie. Within the preceding 4-weeks or so) will influence HbA1c level more than glucose 12 or more weeks ago.

9. METHODS OF ESTIMATION 1. High performance liquid chromatography (HPLC): The HbA1c result is calculated as a ratio to total Hb by using a chromatogram 2. Radiolmmunoassays 3. Enzymrimmuno assays 4. Isoelectric to focusing 5. Colorimetric method 6. Microcoleum ion exchange technique 7. Capillary electrophoresis 8. Boronate afinity chromatography.

10. ADVANTANGES AND DISADVANTAGES OF VARIOUS ASSAY METHODS ASSAYPRINCIPLEADVANTAGESDISADVANTAGES Ion Exchange Chromatograp hy HbA1c has lower isoelectric point and migrate faster than other Hb Componets Can inspect chromogram s for Hb Variants measuremen t with great precision Variable interference from the Haemoglobinopath eis, HbF and Carbamylated Hb but the current ion in exchange assays correct for HbF & carbonlated Hb does not interfere.

11. contd Boronate affinity Glucose binds to m- aminophem lborononic acid Minimal Interferenc e from Haemoglob inopatheis, HbF and Carbamylat ed Hb. Measures not only glucation of N-terminal valine on B chain but also B-chains glycated at other sites and glycated Alpha-chain.

12. contd Immunoassays Antibody binds to glucose and between 4- 10 N- terminal amino acids on b-chain. Not affected by HbE, Hb D, or carbamylate d Hb, Relatively easy to implement under many different format. May be affected by haemoglonopa theis with altered amino acids or binding sites. Some Interferes with HbF

13. PRINCIPLES & PROCEDURES  Using kit of HbA1c.  A haemolysed preparation of whole blood is mixed continously for 5 minutes with a weak binding caton-exchange resin during this time the non-glycated haemoglobin, which consist of the bulk of the haemoglobin (HbAo) binds to the resin. After the mixing period a filter is used to separate the supernatant containing the

14. CONTD.  Glycohaemoglobin from the resin. The percent glycohaemoglobin is determine by measuring the absorbance at 415 nm (405 - 420 nm acceptable) of the glycohaemoglobin fraction and the total haemoglobin fraction. The ration of the two absorbances gives the percent haemoglobin.

15. CONTD.  Using Ghb analyser.  It uses low pressure cation exchange chromatography in conjuction with gradient elution to separate human haemoglobin subtype and variants from haemolysed whole blood. The separated haemoglobin fraction are monitored by means of absorption of light at 415mn the chromatogram obtained is recorded

16. CONTD.  And stored by the on board computer. The analyser performs the analysis of the chromatogram and generate a printed report.

17. FACTORS THAT AFFECTS THE TEST RESULT INCREASEDECREASE Increase in HbF↑ Haemolysis/ble eding ↓ Haemoglobinop atheis (HbS) Glucose -6- Pphosphate dehydrogenase deficency Invalidresult

18. contd Surgery, anaemia, blood transfusion, high erythrocyte turnover InvalidResult Liver disease↓ Blood donation↑ Vit B12 folate deficiency (longer rbc infespan) ↑

19. contd Splenectomy (Increase rbc life-span) ↑ Pregnancy (Hormonal changes) ↑ Renal failure(dialysis) ↓

20. ESTIMATED AVERAGE GLUCOSE (EAG)  This term is used to understand the approximate relationship between HbA1c and glucose Concentration.  Formular: eAG (mg/dl)=(28.7xHbA1c)-46.7  eAG (mmollL) = (1.59 x HbA1c) – 2.59

21. NORMAL RANGE: Varies with different Lab but the reference laboratory value is:- Non-diabetic adult – 2.2-5.0% Diabetic adult: 7% (ADA guideline).

22. RESULT INTERPRETATION Possible meaning of Abnormal result INCREASEDECREASE AlcoholChronic loss of blood HyperghycemiaChronic renal failure Lead poisioningHaemolytic Anaemia Newly diagnose DMSickle-cell anaemia

23. contd Poor DM controlThalassemia Pregnancy

24. RECOMMENDATIONS  Management goals should be aimed at preventing the development of these complications and/or delaying the onset of these complications for a prolonged or healthy life, therefore a test of this nature (HbA1c) is highly recommended for adequate management control of DM.  It should be performed at least twice a year in patients who are meeting treatment goal and who have stable glycerinic control.

25. CONTD.  Quarterly in patient whose therapy has changed or who are not meeting glycemic goals.  As needed to assist with decision-making on therapy changes.  Glycernic goals should be individualized, more stringent value ( less than 6%) reduce complications..

26. CONCLUSION Diabetes is now affecting many in the work place, it has a major and deleterious impact on both individual and national productivity. The socio-economic consequences of diabetes and its complications could have a seriously negative impact in the economics of developed and developing nation.

27. CONTD.  DM is a world health Challenge and the complications such as Retinopathy, neuropathy,Nephropathy, Skin lesions (ulcer), diabetic Keto acidosis can be life threatening and difficult to reverse.

28. REFERENCES:  Oxford hand book of clinical and Laboratory Investigations by Drew Provan & Andrew Krentz (2002).  McGraw-Hills Manual of Laboratory & Diagnostic test (Denis D. Willson 1976).  Blacks Medical Dictionary 41 st Edition (2005)  Huisman T.H, Mantis E.A Dozy A (1958)  Chromatography of Haemoglobin types on Carbozymethylcellulose. Clin. Med. 52 (2): 312 -327  Koenig R.J, Peterson C.M, Jones R.L,( et al 1976 ) “Correlation of glucose regulation and Haemoglobin A1c in diabetes mellitus” N.Engl. J. Med. 295 (8): 417 -420.