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University

of Tabuk- Faculty of Medicine


Laboratory Medicine
Introduction

• Diabetes mellitus is a common


metabolic disorder characterized by
chronic hyperglycemia with
disturbances of carbohydrate, fat
and protein metabolism
• 4th leading cause of death.
• It is estimated that only half of the
patients with diabetes have been
diagnosed.

Global burden of DM, millions


Hormonal Regulation of Blood Glucose Levels:

Glucose increase Glucose decrease


- Glucagon (increase glycogenolysis + - Insulin (transport glucose
gluconeogenesis by liver) across cell membranes)

- ACTH (increase glucocorticoids) - Glucose metabolism

- Glucocorticoids (increase - Glycogen synthesis


gluconeogenesis)
- Epinephrine (similar to glucagon) - Lipogenesis

- Thyroxin (similar to glucagon)


- Growth hormone (insulin
antagonist)
- Somatostatin (decrease insulin
secretion)
Classification of Diabetes Mellitus

1. Type 1 DM.
2. Type 2 DM.
3. Impaired Fasting Glucose (IFG) and Impaired
Glucose Tolerance (IGT).
4. Gestation Diabetes/IGT.
5. Maturity Onset Diabetes of the Young
(MODY).
Criteria for diagnosing DM

FPG ≥ 126 mg/dl


OR
2-h plasma glucose ≥ 200 mg/dl during an OGTT
OR
Hb A1c ≥ 6.5%.
OR
Casual plasma glucose >200 mg/dl with symptoms of diabetes

à NOTE: In asymptomatic patients, two diagnostic tests are required


to confirm diabetes
Type 1 diabetes mellitus

• Type 1 DM is an autoimmune disease.


• There is a strong association with certain HLA
• Environmental triggers:
– Viral antigens (e.g. coxsackie B)
– Proteins in cows’ milk
– Vitamin D deficiency.
• There is a considerable geographic variation
• Islet cell antibodies (ICA) are frequently present in the
plasma + antibodies to insulin + glutamic acid
decarboxylase (GAD) Ab
• β-cell destruction is initiated by activated T-lymphocytes
Type 2 diabetes mellitus

• β-cell inadequate insulin response to hyperglycaemia +


insulin resistance
• Hyperglycaemia (glucotoxicity) + hyperlipidaemia
(lipotoxicity) cause insulin resistance and β-cell dysfunction
• Decreased secretion of incretins have been observed in
patients with type 2 DM
• Type 2 DM shows a strong familial incidence: >50% if both
parents have the condition.
Genetic Defects in Carbohydrate
Metabolism
• These disorders cause Monogenic diabetes (1-2%)
• Clinical features overlap with Type I & I
• Neonatal diabetes: diagnosed within 6m, transient or
permanent (K-channel gene mutation), 90% has good
control with sulphonylureas
• Maturity onset diabetes of the young (MODY): <25y
– Glucokinase mutation: mild hyperglycemia, Rx: Diet
– Hepatic nuclear factor 1 alpha (HNF-1α): Rx:
sulphonylureas
Major Features of Type 1/2 Diabetes Mellitus
and MODY
Clinical features Type I DM Type II DM MODY

Typical age of <25 years >25 years >25 years


diagnosis

Body weight Usually not obese Overweight to obese No obesity

Autoantibodies Present Absent Absent

Insulin Yes No No
dependence

Family history Infrequent Frequent Yes, in multiple


generations

Diabetic High risk Low risk Low risk


ketoacidosis
Measurement of Glucose
Concentration
• Any of the following tests can be used for diagnosis of DM but any test
need confirmation unless symptoms of DM exist:
1. Fasting Plasma Glucose (FPG)
2. Postprandial Glucose
3. Random blood glucose
4. Oral Glucose Tolerance Test (OGTT)
5. Glycylated-Hemoglobin
• Collection tube: The tube contains sodium fluoride to inhibit
glycolysis+ Potassium oxalate as an anticoagulant
• Plasma glucose concentration tends to be 10–15% > whole blood ?
• The arterial blood glucose are 20mg/dl > venous blood.
• Glycolytic activity of WBCs & RBCs decreases glucose concentration by 7%/
1h.
Methods of Glucose Measurement
• The Hexokinase method

• The Glucose oxidase method

• The Glucose Dehydrogenase method


Accuracy of glucose meters
• Blood glucose concentrations are now frequently
measured using glucometers (Glucocheck)
• The following criteria must be met by a glucose home
monitoring method:
1. Glucose meter should not be used for diagnosis of DM

2. Food and Drug Administration (FDA) criteria:


– At a glucose concentration <75 mg/dL: 95% of all values should
fall within +/-15 mg/dL of the glucose value obtained by a
clinical laboratory- based reference method
– For glucose values >75 mg/dL, 95% of the individual values must
fall within +/-20% of the glucose value determined by a
reference method.

3. ADA criteria suggest that the glucose value measured by


a glucose meter should be within +/-5% of the value
obtained by a laboratory-based glucose assay.
Fasting Plasma Glucose (FPG)

• Assess the capability of basal levels of insulin to control


glycogenolysis and gluconeogenesis.
• Determination of fasting plasma glucose is the very first
procedure to be performed when entertaining a diagnosis of
diabetes.
• Patient preparation:
Ø The patient is kept on an average usual diet and, after an
overnight fast (no food or sweetened drinks after midnight),
blood is drawn in the morning → Not less than 8hrs of fasting.
Glucose Tolerance and 2-hour
Postprandial Tests
Indications Procedure
Equivocal fasting/random glucose 1. Normal diet, containing at least 250g
CHO/day for 3 days
Unexplained glycosuria, particularly in 2. Fast overnight
pregnancy
Clinical features of DM or its 3. Fasting basal glucose
complication with normal blood glucose
4. Give 75g glucose in water, take further
blood sample after 2 hours
Patient should rest throughout the
procedure, water is allowed, smoking is
prohibited.

• If a patient vomits, the test must be repeated.


• In pregnant women, the test dose is 100 g glucose.
• In patients with carbohydrate malabsorption, an intravenous glucose tolerance
test is used with different reference ranges.
Glycated Hemoglobin(Hba1C )
• During RBCs life span Hb A and other forms become glycated
(glucose reacts with β-polypeptide N-terminal valines) in a
degree proportional to the level of glucose.
• Hba1C is a form of HB that is measured to identify the average
glucose concentration over previous 2-3 m as this is the life span
of RBCs
• Useful in the diagnosis and monitoring (assessment of the efficacy of
long-term therapeutic control of diabetic patients) of Type II DM
Measurement of HbA1C
• Sample: EDTA whole blood
• Methods
1. Structural differences based
– Immunoassay (point of care)
– Affinity chromatography
2. Charge differences based
– Ion exchange chromatochragy
– Isoelectric focusing
– High pressure liquid chromatography

Normal: Less than 6.5


Excellent: 6.5-7.5
Good: 7.5-8.5
Fair: 8.5-9.5
Poor: Greater than 9.5
Situations where HBA1c is not appropriate for
the diagnosis of Diabetes
Children
Type 1 diabetes
<2 months symptoms of DM
Medications causing rapid glucose rise: steroids
Patients with acute pancreatic damage
Pregnancy
Abnormal red cell survival (as in hemolytic anemia and
thalassemia) à lower Hb A1c values
Abnormal variants of HB (Hb S, Hb C)
Splenectomy and polycythemia à increase Hb A1c values
Criteria for Testing for Pre-diabetes
and Diabetes
• Impaired glucose tolerance is not a clinical entity in itself, but
defines a risk category for progression to diabetes.
• Impaired fasting glycaemia is a further category of abnormal
glucose tolerance
• Some individuals found to be diabetic on OGTT; others have
IGT, eventually develop DM
• Patients with IGT should be given dietary and lifestyle advice
and reviewed regularly.
• Have a similar predisposition to MI and stroke as patients with
frank diabetes, but they are not at increased risk of
microvascular complications.
Criteria for metabolic syndrome include
≥3 of the following risk factors:
1. Central obesity (waist circumference: ≥40 inches in men,
≥35 inches in women).
2. Insulin resistance: Fasting glucose >100 mg/dL.
3. Elevated triglycerides ( >150 mg/dL).
4. Reduced high density lipoprotein cholesterol (HDL
cholesterol: <40 mg/dL for men, <50 mg/dL for women).
5. Elevated blood pressure (>130 mm of Hg for systolic, or >85
mm of Hg for diastolic) or drug treatment for hypertension.
Criteria for the testing and diagnosis
of gestational diabetes
• Gestational diabetes is diabetes or IGT with onset or first
recognized during pregnancy.
• Pregnant patients with any degree of glucose intolerance
should be managed as if they have diabetes.
• Pregnancy decreases glucose tolerance, and patients with
gestational diabetes may revert to a normal glucose tolerance
post-partum.
Hyperglycemia in non diabetic

• Pancreatectomy
• Acromegaly
• Cushing syndrome .
• Glucoganoma.
• Primary aldosteronism.
• Severe stress.
• Hyperthyroidism.
• Drugs (thyroxin, phenytoin)
Glycosuria
• It is the excretion of glucose in urine. Appears when:
→ Blood glucose exceeds the renal threshold for
reabsorption.
• Normally lies at about 170 mg/dL, but it may be:
– Lower in renal tubular disease.
– Elevated in diabetics to above 300 mg/dL.
• High glucose concentration increases the urinary specific
gravity.
• Glycosuria usually measured by semi-quantitative tests +1,
+2… (As in routine urinalysis). Quantitative urinary glucose
determinations are seldom necessary (usually between 0.5 to
2.0 g/dL).
Non-diabetic causes of glycosuria
Glycosuria not associated with hyperglycemia ( due to decreased
renal reabsorption):
• Pregnancy.
• Fanconi’s syndrome.
• Renal glycosuria (true renal diabetes).
• Nephrotic syndrome.

Glycosuria associated with hyperglycemia:


• Thyrotoxicosis.
• Cushing’s syndrome.
• Damage to CNS.
• Infections.
• Anesthesia.
• Excitement and stress.
Ketones
• Produced in the liver through FA metabolism
• Mainly seen in type 1 DM , rarely type 2.
– Acetone 2%
– Acetoacetic acid 20%
– 3-β-hydroxybutyric acid 78%
Ketones Measurement
• Specimen: fresh sample, stoppered and analyzed
immediately
• Gerhardt`s: historical test
FeCl3+ acetoacetic acid= red color
• Striptest (Sodium nitroprusside):
NaFe[CN]5CN+ acetoacetic acid (alkaline)=
purple color (+glycerin measures acetone also)
• Enzyme method (automated):
3-hydroxybutyrate dehydrogenase
• In severe ketoacidosis we can measure the anion
gap and the lactic acid value to assess the severity.
Determination of Insulin and C-peptide

• C-peptide and insulin are not used for the initial diagnosis of diabetes.
• Proinsulin à Insulin + C-peptide .
• t1/2 of C-peptide is longer than that of insulin.
• Serum insulin and C-peptide are measured by radioimmunoassay to:
1. Differentiate between MODY disease and type 1 DM (very low level of C-
peptide in type 1 DM)
2. Measure the insulin secretory capacity in individuals who had developed
antibodies to the hormone following treatment with non-human insulin,
since C-peptide do not react with insulin antibodies → (C-peptide >>
insulin level).
Microalbuminuria
• It is a persistent albuminuria 2/3 of urine samples
within 3-6 months:
– 20-200μg/min or 30-300mg/24h or 4h overnight
– Albumin-creatinine ratio= ≥300mg/24h, >200μg/min, or ≥300μg/mg
• DM cause progressive insult to the kidney ultimately
results over years in diabetic nephropathy
• Annual measurement is recommended
• Diabetic lead to increased urinary excretion and
clearance of proteins with a MW >40,000→ this can
become non-selective for the larger proteins and may
progress to several grams of protein loss/24 hour.
Anti-Insulin Antibodies
In the past beef or pork insulin were used in DM treatment → IgG
antibodies was demonstrated in nearly all patients after few weeks.
• The extent of antibody formation is a function of:
(1) Immune response.
(2) Insulin source (beef is more immunogenic than pork).
(3) Insulin type (long-acting preparations are more immunogenic than
regular).
(4) Purity of the insulin preparation.
• The clinical significance is not completely known (Increased titers of IgG
antibodies directed against beef insulin may be associated with
resistance).
• With the currently increasing use of purified pork insulin and human
insulin preparations, problems of immunologic resistance to insulin are
quickly disappearing.