Diabetic Profile - Lab Diagnosis

Sri Lanka Society for Medical Laboratory Science
slsmls.org / kumudeshr@gmail.com
Diabetic Profile
Ravi Kumudesh
MSc/BSc/Dip(MLT)

Diabetes Mellitus
• It is a chronic disease due to
disorder of carbohydrate
metabolism, due to insulin
deficiency results in
hyperglycemia (increased
blood glucose level) &
glucourea (presence of
glucose in urine).
• Associated with several
changes in metabolism; such
as metabolism of proteins &
fats.

• Glucosuria.
• Large volume of urine
• increase urination frequency
(Polyuria)
• Polyphagia (eats more frequently)
• Several metabolic changes

Metabolic changes in diabetes
Include increase in:
 Fat catabolisim leads to increase in FFAs in blood & liver.
 Acetyl.coA leads to increase formation of cholesterol &
risk of atherosclerosis.
 ketone bodies generation in blood and urine leads to
acidosis.
 catabolism of tissue protein due to energy requirement
(because glucose can't uptake by cells) lead to weight
loss and increase in level of amino acids in blood & more
formation of urea by deamination of amino acid.

Types of diabetes
• Type I diabetes mellitus (TIDM)
• Type 2 diabetes mellitus (TIIDM)
• Gestational diabetes mellitus (GDM)
• Other "due to drugs or chemicals"

Is group of tests that are used to diagnose diabetes
or its complications , it includes:
 Blood glucose
4 types: FBS, PPBS, RBS, OGGT
 Urine Analysis
Urine Sugar / Urine Protein /Urine Microalbumin / Ketones
 HbA1C
 Insulin
 ICA (islent cell antibody) for type I
 C-peptide

Urine Analysis

1. Urine Sugar
Detection of urinary glucose (Glucosuria)

Glucosuria
o First-line screening test for diabetes
mellitus
o Normally glucose does not appear in urine
until the plasma glucose rises above 160-
180 mg/dl.
o In certain individuals due to low renal
threshold glucose may be present despite
normal blood glucose levels.
o Conversely renal threshold increases with
age so many diabetics may not have
Glycosuria despite high blood sugar levels.
Positive
Benedict’s test

o A specific and convenient method to
detect glucosuria is the paper strip
impregnated with glucose oxidase and
a chromogen system (Clinistix,
Diastix), which is sensitive to as little
as 0.1% glucose in urine.
o Diastix can be directly applied to the
urinary stream, and differing color
responses of the indicator strip reflect
glucose concentration.
o Benedict’s and Fehling’s test can
also detect glucosuria.
Diastix-
Reagent strips

Microalbuminuria
2. Urine Microalbumin

o The importance of micro-
albuminuria in the diabetic
patient is that it is a signal
of early reversible renal
damage.
o Performing an albumin-to-
creatinine ratio is probably
easiest.
o Microalbuminuria is a
common finding (even at
diagnosis) in type 2
diabetes mellitus and is a
risk factor for macro
vascular (especially
coronary heart) disease.
Gradation of turbidity is linked to
protein concentration
Microalbuminuria

Microalbuminuria
o May be defined as an albumin
excretion rate intermediate
between normality (2.5-25 mg/day)
and macroalbuminuria
(250mg/day).
o The small increase in urinary
albumin excretion is not detected
by simple albumin stick tests and
requires confirmation by careful
quantization in a 24 hr urine
specimen.

Qualitative
 Dipstick method
Quantitative
Assays for Microalbuminurea
 Enzyme linked Immunosorbant assay
 Radioimmuno assay
 Immunoturbidometric assay

Specimen Collection for Microalbimin
• Collect freshly voided urine in a clean, dry
container
• Preservatives should be avoided
• Samples which cannot be tested within 3 days of
collection should be refrigerated
• Samples should not be frozen
• The test should be free from significant
interference from glucosuria, pH, ketonuria or
bacterial contamination

MICRAL Strips
 Micral strip screening tests offer a cost-
effective method of screening
 Dip sticks show acceptable sensitivity (95%)
and specificity (93%)
 All positive tests should be confirmed by
more specific methods

False Positives
 Hyper filtration (Newly diagnosed diabetes)
 Exercise
 Marked hypertension
 Congestive Heart Failure
 Urinary Tract Infection
 Acute febrile illness

3. Urine Ketone Bodies
Ketonuria

What Are Ketones?
 Acids that result when the body does not have
enough insulin and uses fats for energy
 May occur when insulin is not given, during
illness or extreme bodily stress, or with
dehydration
 Can cause abdominal pain, nausea, and vomiting
 Without sufficient insulin ketones continue to
build up in the blood and result in diabetic
ketoacidosis (DKA)

Why Test for Ketones?
 DKA is a critical emergency state
 Early detection and treatment of ketones
prevents diabetic ketoacidosis (DKA) and
hospitalizations due to DKA
 Untreated, progression to DKA may lead to
severe dehydration, coma, permanent brain
damage, or death
 DKA is the number one reason for hospitalizing
children with diabetes

When Should Ketones Be Checked?
The DMMP should specify, generally:
 When blood glucose remains elevated
 During acute illness, infection or fever
 Whenever symptoms of DKA are present
 Nausea
 Vomiting or diarrhea
 Abdominal Pain
 Fruity breath odor
 Rapid breathing
 Thirst and frequent urination
 Fatigue or lethargy
 Common symptoms including fruity odor to breath,
nausea, vomiting, drowsiness, abdominal pain
−

How Quickly Does DKA Progress?
 An isolated high blood glucose reading, in the absence
of other symptoms is not cause for alarm
 DKA usually develops over hours, or even days
 DKA can progress much more quickly for students who
use insulin pumps, or those who have an illness or
infection
 Most at risk when symptoms of DKA are mistaken for
flu and high blood glucose is unchecked and untreated

Checking for Ketones
 Urine testing
 Most widely used method
 Blood testing
 Requires a special meter and strip
 Procedure similar to blood glucose checks

1. Gather supplies
2. Student urinates in clean cup
3. Put on gloves, if performed by someone
other
than student
4. Dip the ketone test strip in the cup
containing urine. Shake off excess urine
5. Wait 15 - 60 seconds
6. Read results at designated time
7. Record results, take action per DMMP
How to Test Urine Ketones

Test Results: Color Code
 no ketones
 trace
 small
 moderate
 large ketones present

Considerations
 Colors on strips and timing vary according
to brand
 If using a scale with “urine glucose” and
“urine ketones,” be sure to read the correct
scale when testing for ketones
 Follow package instructions regarding
expiration dates, time since opening,
correct handling, etc., as incorrect results
may occur

How To Test for Blood Ketones
1. Prepare lancing device
2. Wash hands using warm soapy
water and dry them completely
3. Remove the test strip from its foil
packet
4. Insert the three black lines at the
end of the test strip into the strip
port
5. Push the test strip in until it stops

How To Test for Blood Ketones
6. Touch the blood drop to the purple
area on the top of the test strip. The
blood is drawn into the test strip
7. Continue to touch the blood drop to
the purple area on the top of the test
strip until the monitor begins the test
8. The blood ß-Ketone result shows on
the display window with the word
KETONE

Ketonuria
o Qualitative detection of
ketone bodies can be
accomplished by nitroprusside
tests (Acetest or Ketostix),
Rothera’s test etc.
o These tests do not detect
Beta-hydroxy butyric acid,
which lacks a ketone group
o Ketone bodies may be
present in a normal subject as
a result of simple prolonged
fasting.
Positive
Rothera’s
test
Ketostix-
Reagent
strips

Blood Glucose Levels

1. Fasting blood sugar (FBS)
• Measures blood glucose after fasting for at least 8-12 hrs
• It often is the first test done to check for diabetes.
• patient with mild or borderline diabetes may present
with normal FBG values.
• If diabetes is suspected, GTT can confirm the diagnosis.
Normal levels:
70-110mg/dl

2. Post-Prandial Blood Sugar (2-hour PPBS)
• After the patient fasts for 12 hours, a meal is
given which contains starch and sugar
(approx. 100 gm).
• Then after 2 hours blood is collected to
measure glucose level.
• home blood sugar test is the most common
way to check 2-hour postprandial blood sugar
levels.

3. Random blood sugar (RBS)
 measures blood glucose randomly at any time
throughout the day without patient fasting.
 it is useful because glucose levels in healthy
people don’t vary widely throughout the day.
 blood glucose levels that vary widely may
indicate a problem.

4. Oral glucose tolerance test (OGTT)
• Glucose Tolerance is defined as the capacity of the body
to tolerate an extra load of glucose or it measures the
body's ability to use glucose.
• It is series of blood glucose measurements taken after
drink glucose liquid
• It is considered as definitive diagnostic test for DM.
• It is ordered to:
Confirm the diagnosis, in pre-diabetic
Diagnose gestational diabetes (most commonly)
• Recommended if 100-126 mg/dL (5.5 mmol/L-7.0 mmol/L)

 Arrive FBS: After an overnight fasting
(10-12 hrs)
 Drink: 75-100g dissolved in 250-300ml of
water and given orally.
 After drink: blood samples and urine are
collected every 30min for 3hrs
(1 hr, 1.5 hr , 2hr, 2.5hr, 3hr )
 A curve between time and blood glucose
concentration, is plotted.
Procedure

• Extended GTT :
Glucose measured for 4-5 hrs after giving glucose to see how the
curve behaves below the normal fasting glucose limits. Done in
some conditions causing hypoglycaemia.
• Cortisone Stressed GTT :
Can be used for detecting latent DM.
• Intravenous GTT :
 Is done if oral glucose is not tolerated or oral GTT curve is
flat.
 In these cases 20% glucose as 0.5g glucose/Kg body weight.
 Usually peak occurs within 30 min after infusion and returns
to normal after 90 min.
Other types of OGTT

Interpretation
 Normal Response :
FBS is normal. After 1 hr it will rise, returns to normal fasting
level within 2 hours.
 Diabetic curve :
FBS: 140mg/dl or 7.8 mmol/L. After 2 hr: 200mg/dl (11
mmol/L) or more. Glucosuria is usually seen
 Impaired GTT:
 with 2hrs glucose level between 140mg/dl - 200mg/dl
 It is not abnormal but must be followed up for DM.

Hypoglycemia :
• When blood glucose falls below 60 mg/dl.
Causes
1. Most commonly seen in overdose of insulin in
treatment of DM.
2. Hypothroidism.
3. Insulin secreting tumours of pancrease – rare.
4. Hypoadrenahsm (Addison's disease)
5. Hypopitruitism.
6. Severe exercise.
7. Starvation.

Principle:
Glucose + H2O + O2 Gluconic acid + H2O2
2H2O2 + 4 aminoantipyrine + PHBS Quinoneimine dye + H2O
Red color
POD
GOD

Kit components
• Glucose Oxidase Reagent :
mixture of:
glucose oxidase + peroxidase+ aminoantipyrine+ buffer
• Glucose standard Reagent :
conc. 100mg/dl or 5.55 mmol/L

Procedure
SampleStandardReagent blank
1ml1 ml1 mlGlucose oxidase reagent
10 µl-Sample (serum)
-10 µl-Glucose standard
• Prepare the reaction as the following:
• Mix, incubates at 37oC for 10min
• Read abs at 510nm

Calculation
Glucose conc. = Abs. Sample X Conc. Standard
Abs. Standard ..

CBC – Capillary Blood Glucose

Special Tests

1. Glycohaemoglobin
Hb A1C

Hb A1C
 HbA1C: is glucose bound to hemoglobin
 Measures blood glucose conc. over a longer period of time
 It indicates how well diabetes has been controlled in the 2-3
months before the test.
 The A1C level is directly related to complications from diabetes
 Type of sample: whole blood in EDTA tube
Normal Values
 Glycohemoglobin A1c:4.5%-5.7%
 Total glycohemoglobin:5.3%-7.5%

Key Messages
1. Glycated hemoglobin (A1C)  measure every
3 months (6 months if stable at target)
2. Self monitoring Blood Glucose (SMBG) is an aid
to assess interventions and hypoglycemia
3. Individualize the frequency of SMBG
4. SMBG and continuous glucose monitoring (CGM)
needs to be linked with structured educational
program to facilitate behavior change

Glycated Hemoglobin: A1C
• Reliable measure of mean plasma glucose
over 3-4 months
• Valuable indicator of treatment effectiveness
• Measure every 3 months when glycemic
targets are not being met or treatments
adjusted
• Measure every 6 months if stable at glycemic
targets

Conditions that can Affect Value
Factors affecting A1C Increased A1C Decreased A1C Variable Change
in A1C
Erythropoiesis B12/Fe deficiency Decreased
erythropoiesis
Use of EPO, Fe, or B12
Reticulocytosis
Chronic liver Dx
Altered hemoglobin Fetal hemoglobin
Hemoglobinopathi
es Methemoglobin
Altered glycation Chronic renal failure
↓↓erythrocyte pH
ASA, vitamin C/E
Hemoglobinopathies
↑ erythrocyte pH
Erythrocyte destruction Splenectomy Hemoglobinopathies
Chronic renal failure
Splenomegaly
Rheumatoid arthritis
HAART meds,
Assays Hyperbilirubinemia
Carbamylated Hb
ETOH
Chronic opiates
Hypertriglyceridemia

2. Insulin Levels

 Insulin is the primary hormone responsible for controlling glucose
metabolism, and its secretion is governed by plasma glucose
concentration.
 The insulin molecule is synthesized in the pancreas
 The principal function of insulin is to control the uptake and
utilization of glucose in the peripheral tissues.
 Insulin concentrations are severely reduced in insulindependent
diabetes mellitus (IDDM) Other conditions, non-insulin-dependent
diabetes mellitus (NIDDM), obesity, and some endocrine
dysfunctions.
Insulin Test - Clinical Relevance

 The Insulin ELISA is a two-site enzyme immunoassay utilizing the direct
sandwich technique with two monoclonal antibodies directed against separate
antigenic determinants of the insulin molecule.
 Specimen, control, or standard is pipetted into the sample well, then followed
by the addition of peroxidase-conjugated anti-insulin antibodies.
 Insulin present in the sample will bind to anti-insulin antibodies bound to the
sample well, while the peroxidase-conjugated anti-insulin antibodies will also
bind to the insulin at the same time.
 After washing to remove unbound enzyme-labelled antibodies, TMB-labelled
substrate is added and binds to the conjugated antibodies.
 Acid is added to the sample well to stop the reaction, and the colorimetric
endpoint is read on a microplate spectrophotometer set to the appropriate
light wavelength.
Insulin - Test Principle

3. Islet Cell Antibody (ICA)

 Using the indirect fluorescent antibody method enables serologic
assessment or possible detection of pancreatic disease.
 The presence of a (histologically defined) circulating antibody to one or
more of the islet cell antigens can aid in patient diagnosis and prognosis.
 The substrate utilized in this kit is sections of monkey pancreas. Islet Cell
antibodies have been associated with a group of "autoimmune" endocrine
disorders, more specifically with insulin dependent diabetes.
 Organ-specific autoimmunity is characterized by the presence of
antibodies in patients that can be detected years before the onset of the
clinical symptoms.
 Patients with autoimmune thyroiditis, adrenalitis or gastritis have an
increased risk of developing insulin dependent diabetes at any age.
Demonstration of Islet Cell Antibody (ICA)

 The indirect fluorescent antibody test is used for the detection of human
IgG antibody to the antigens of monkey pancreas islet cells.
 Tissue is placed in the wells of specially prepared microscope slides.
 Dilutions of patient sera are placed on the wells where antibody, if present,
binds to the antigen.
 The reaction is visualized through the use of a conjugate.
 The conjugate is fluorescein isothiocyanate (FITC) labeled, anti-human IgG
 Excitation of the FITC by ultraviolet (UV) light causes this dye to emit
longer, visible, wavelengths of light in the yellow-green portion of the color
spectrum.
 The conjugate will bind with human IgG antibodies attached to the
antigens causing fluorescence when viewed through a microscope
equipped with a UV light source
Test Principle and Procedure

4. C-peptide Test

 C-peptide testing can be used for a few different
purposes
 C-peptide is a substance produced by the beta cells in
the pancreas when pro insulin splits apart and forms
one molecule of C-peptide and one molecule of insulin
 Insulin is the hormone that is vital for the body to use
its main energy source, glucose
 Since C-peptide and insulin are produced at the same
rate, C-peptide is a useful marker of insulin production
C-peptide Test

 Sweating
 Palpitations
 Hunger
 Confusion
 Blurred vision
 Fainting
 In severe cases, seizures
 loss of consciousness
When is it ordered?

What does the test result mean?
 A high level of C-peptide generally indicates a high level
of endogenous insulin production. This may be in response to a high blood
glucose caused by glucose intake and/or insulin resistance.
 A high level of C-peptide is also seen with insulinomas and may be seen
with low blood potassium, Cushing syndrome, and renal failure
 When used for monitoring, decreasing levels of C-peptide in someone with
an insulinoma indicate a response to treatment; levels that are increasing
may indicate a tumor recurrence
 A low level of C-peptide is associated with a low level of insulin production.
This can occur when insufficient insulin is being produced by the beta cells,
with diabetes for example, or when production is suppressed by treatment
with exogenousinsulin

Not Only Test, But ….?

Diabetic Profile - Lab Diagnosis

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