Role of the Laboratory in Differential Diagnosis of Diabetes Mellitus

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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 Glucose concentration (mmol I-1) Venous sampling Capillary sampling Whole blood Plasma Whole blood Plasma Glucose concentration (mmol I-1) Venous sampling Capillary sampling Whole blood Plasma Whole blood Plasma Glucose concentration (mmol I-1) Venous sampling Capillary sampling Whole blood Plasma Whole blood Plasma 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
lt7.8 8.9-12.2 lt6.7 7.8-11.1 lt7.8 7.8-11.1 lt6.7 6.7-10.0 Impaired glucose tolerance Fasting sample 2 h after glucose load
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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
Interference Measurement Method
Carbamyl Hb HbF Temperature- sensitive HbA1c Cation exchange
HbA1c Monoclonal antibody
Glycated Hb Affinity chromatography Phenyl boronate matrix Latex agglutination Fluorescence quenching