1 Role of the Laboratory in Differential Diagnosis of Diabetes Mellitus Dr. Essam H. Jiffri

2 INTRODUCTION -The demonstration of hyperglycemia or hypoglycemia under specific conditions is used to diagnose diabetes mellitus and hypoglycemic conditions. -Other laboratory tests have been developed to identify insulinomas and to monitor glycaemic control and the development of renal complications.

3 Glucose Estimation -Glucose may be estimated in either plasma or whole blood. -The glucose concentration in whole blood is approximately 15% lower than the glucose concentration in serum or plasma, because the volume of distribution of glucose is lower, as erythrocytes contain less free water than plasma. -Samples for glucose can be obtained either by veinpuncture or by a fingerprick technique (collected in capillary tubes).

4 Glucose Estimation -Blood cells continue to metabolize glucose after veinpuncture and serum or plasma must be refrigerated and separated from the cells within 1 hour to prevent substantial losses of glucose by the cellular fraction. -A preservative that inhibits glycolysis should be used (sodium fluoride, together with potassium oxalate as an anticoagulant, is used for this purpose).

5 Glucose Estimation -Test strips which measure blood glucose can be useful in obtaining an indication of blood glucose concentrations, but diagnosis should be based on laboratory measurements.

6 Fasting Plasma Glucose -A more important measurement is the fasting glucose concentration, which is drawn after an overnight fast (10-16 h). -A fasting glucose concentration greater than 140 mg/dL (7.8 mmol/L) is considered diagnostic for diabetes mellitus by the National Diabetes Data Group.

7 Two-Hour Postprandial Plasma Glucose -The two-hour postprandial glucose measurement is often used in conjunction with the fasting plasma glucose. -The patient is advised to consume a meal that contains approximately 75 grams of carbohydrates.

8 Two-Hour Postprandial Plasma Glucose -Two hours after eating, a blood sample is drawn for plasma glucose measurement. -A glucose value greater than 200 mg/ dl (11.1 mmol/L) indicates diabetes mellitus.

9 Oral Glucose Tolerance Test (OGTT) -The OGTT is the most sensitive test for the diagnosis of diabetes. -A sample of the patient's blood is drawn after an over night fast. -The patient then consumes 75g of a glucose solution and blood is drawn every 30 minutes for two hours.

10 Oral Glucose Tolerance Test (OGTT) -For children, glucose is administered at glucose/kg body weight to a 75 g maximum. -A plasma glucose greater than or equal to 200 mg/dL (11.1 mmol/L) at the 2-hour time point indicates diabetes mellitus.

11 Oral Glucose Tolerance Test (OGTT) -Impaired glucose tolerance is diagnosed with a plasma glucose between 140 and 200 mg/dL (7.8 and 11.1 mmo1/L) at 2 hours time point in the test. -Gestational diabetes is considered present when the values of the OGTT are greater than the following; fasting, 105 mg/dL (5.8 mmo1/L); 1 h, 190 mg/dl (10.6 mmo1/L), and 2 h, 165 mg/dL (9.2 mmo1/L).

12 Diagnostic criteria for diabetes mellitus and impaired glucose tolerance Glucose concentration (mmol I- 1 ) Venous sampling Capillary sampling Whole blood Plasma Whole blood Plasma ≥ 7.8 ≥ 12.2 ≥ 6.7 ≥ 11.1 ≥ 7.8 ≥ 11.1 ≥6,7 ≥ 10.0 Diabetes mellitus Fasting sample 2 h after glucose load < < < < Impaired glucose tolerance Fasting sample 2 h after glucose load

13 Urinary Glucose -Glucose can be detected in urine using the specific test strips that contain glucose oxidase, peroxidase, and a chromagen. -Other carbohydrates using Benedict's and Febling's reagents.

14 Urinary Ketones -Acetone and acetoacetic acid can be detected in urine using the Acetes TM or Ketostix TM systems. -These tablets or strips use nitroprusside (sodium nitroferricyanide) to detect ketones.

15 Urinary Ketones -Because beta-hydroxybutyric acid lacks a ketone group is not detected by this assay. -Quantitative assays for acetoacetate and beta-hydroxybutyric acid are available using beta-hydroxybutyrate dehydrogenase and either NADH or NAD.

16 Urinary Ketones -If NAD is used as the cofactor and the reaction is buffered at around pH 9.0, beta-hydroxyburyric acid is measured. -On the other hand, a separate reaction using NADH and buffered around pH 7.0 would measure acetoacetic acid.

17 Glycosylated Proteins and HbA 1c -Long-term blood glucose regulation can be followed by measurement of glycosylated haemoglobins, this provides the clinician with a time average picture of the patient's blood glucose concentration.

18 Glycosylated Proteins and HbA 1c -Many proteins are known to react with carbohydrates at the peptide N-terminus forming glycosylated peptides. -Glucose can rapidly react with hemoglobin to form a labile aldimine (Schiff base).

19 Glycosylated Proteins and HbA 1c -The keto amine product is stable and cannot revert back to hemoglobin and glucose. - HbA 1c is the largest subfraction of normal HbA in both diabetic and non-diabetic subjects and is formed by the reaction of the-beta chain of HbA With glucose.

20 Glycosylated Proteins and HbA 1c -The ketoamine (HbA 1c ) fraction reflects the concentration of glucose present in the body over a prolonged time period. -The measurement of glycated haemoglobin therefore gives an indication of the overall degree of blood glycaemic control, in contrast to glucose measurements which give information for a single time-point.

21 Microalbuminuria -Diabetes mellitus causes progressive changes to the kidneys and ultimately results in diabetic renal nephropathy. -This complication progresses over a period of years and may be delayed by aggressive glycaemic control. -An early sign that nephropathy is occurring is an increase in urinary albumin.

22 Microalbuminuria -Microalbumin measurements are useful to assist in diagnosis at an early stage and prior to the development of proteinuria. -Microalbumin concentrations are between 20 to 300 mg/d. -Proteinuria is typically greater than 0.5 g/d.

23 Proteinuria in Diabetes - Many people excrete small quantities of protein in urine, typically around 10 mg/day of mainly low molecular weight proteins such as albumin. -Some diabetic patients develop albumin excretion rates 30 µg/min this range classed as microalbuminuria.

24 METHODS FOR THE DETERMINATION OF GLUCOSE The most used methods of glucose analysis employ the enzymes glucose oxidase or hexokinas. A) Glucose Oxidase B) Hexokinase

25 SELECTED METHODS FOR THE MEASUREMENTS OF GLYCATED HAEMOGLOBINS InterferenceMeasurementMethod Carbamyl Hb HbF Temperature- sensitive HbA 1c Cation exchange HbA 1c Monoclonal antibody Glycated HbAffinity chromatography Phenyl boronate matrix Latex agglutination Fluorescence quenching