1 Role of the Laboratory in Differential Diagnosis of Diabetes Mellitus Dr. Essam H. Jiffri.
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Transcript of 1 Role of the Laboratory in Differential Diagnosis of Diabetes Mellitus Dr. Essam H. Jiffri.
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Role of the Laboratory in Differential Diagnosisof Diabetes Mellitus
Dr. Essam H. Jiffri
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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.
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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).
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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).
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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.
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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.
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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.
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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.
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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.
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Oral Glucose Tolerance Test (OGTT)
-For children, glucose is administered at 1.75 9 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.
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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).
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Diagnostic criteria for diabetes mellitus and impaired glucose tolerance
Glucose concentration (mmol I-1)
Venous sampling Capillary sampling
Whole blood Plasma Whole blood Plasma
Diabetes mellitus
Fasting sample
2 h after glucose load
≥6,7
≥ 10.0 ≥ 7.8
≥ 11.1
≥ 6.7
≥ 11.1 ≥ 7.8
≥ 12.2
Impaired glucose tolerance
Fasting sample
2 h after glucose load
<6.7
6.7-10.0
<7.8
7.8-11.1 <6.7
7.8-11.1 <7.8
8.9-12.2
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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.
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Urinary Ketones
-Acetone and acetoacetic acid can be detected in urine using the AcetesTM or
KetostixTM systems.
-These tablets or strips use nitroprusside (sodium nitroferricyanide) to detect ketones.
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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.
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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.
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Glycosylated Proteins and HbA1c
-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.
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Glycosylated Proteins and HbA1c
-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).
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Glycosylated Proteins and HbA1c
-The keto amine product is stable and cannot revert back to hemoglobin and glucose.
- HbA1c 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.
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Glycosylated Proteins and HbA1c -The ketoamine (HbA1c) 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.
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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.
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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.
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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.
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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
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SELECTED METHODS FOR THE MEASUREMENTS OF GLYCATED HAEMOGLOBINS
Method Measurement Interference
Cation exchange HbA1c Carbamyl Hb
HbF
Temperature-
sensitive
Monoclonal antibodyHbA1c
Affinity chromatography
Phenyl boronate matrix
Latex agglutination
Fluorescence quenching
Glycated Hb