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Sri Lanka Society for Medical Laboratory Scienceslsmls.org / [email protected]
Diabetic Profile
Ravi KumudeshMSc/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.
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• Glucosuria.
• Large volume of urine
• increase urination frequency (Polyuria)
• Polyphagia (eats more frequently)
• Several metabolic changes
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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 toacidosis.
catabolism of tissue protein due to energy requirement(because glucose can't uptake by cells) lead to weightloss and increase in level of amino acids in blood & moreformation of urea by deamination of amino acid.
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Types of diabetes
• Type I diabetes mellitus (TIDM)
• Type 2 diabetes mellitus (TIIDM)
• Gestational diabetes mellitus (GDM)
• Other "due to drugs or chemicals"
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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
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Urine Analysis
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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
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
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Microalbuminuria
Microalbuminuria
oMay be defined as an albumin excretion rate intermediate between normality (2.5-25 mg/day) and macroalbuminuria(250mg/day).
oThe 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
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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
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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
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False Positives
Hyper filtration (Newly diagnosed diabetes)
Exercise
Marked hypertension
Congestive Heart Failure
Urinary Tract Infection
Acute febrile illness
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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)
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Why Test for Ketones?
DKA is a critical emergency state
Early detection and treatment of ketonesprevents 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
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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
−
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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
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Checking for Ketones
Urine testing
Most widely used method
Blood testing
Requires a special meter and strip
Procedure similar to blood glucose checks
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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
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Test Results: Color Code
no ketones trace small moderate
large ketones present
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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
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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
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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
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Ketonuria
o Qualitative detection of ketone bodies can be accomplished by nitroprussidetests (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
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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
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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.
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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.
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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)
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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
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• 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
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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.
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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.
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Principle:
Glucose + H2O + O2 Gluconic acid + H2O2
2H2O2 + 4 aminoantipyrine + PHBS Quinoneimine dye + H2O
Red color
POD
GOD
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Kit components
• Glucose Oxidase Reagent :
mixture of:
glucose oxidase + peroxidase+ aminoantipyrine+ buffer
• Glucose standard Reagent :
conc. 100mg/dl or 5.55 mmol/L
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Procedure
Sample Standard Reagent blank
1ml 1 ml1 ml Glucose 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
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Calculation
Glucose conc. = Abs. Sample X Conc. Standard
Abs. Standard ..
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CBC – Capillary Blood Glucose
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Special Tests
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1. GlycohaemoglobinHb A1C
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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%
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Key Messages1. 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
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Glycated Hemoglobin: A1C
• Reliable measure of mean plasma glucose over 3-4 months
• Valuable indicator of treatment effectiveness
• Measure every 3 months when glycemictargets are not being met or treatments adjusted
• Measure every 6 months if stable at glycemictargets
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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 B12ReticulocytosisChronic liver Dx
Altered hemoglobin Fetal hemoglobin Hemoglobinopathies Methemoglobin
Altered glycation Chronic renal failure ↓↓erythrocyte pH
ASA, vitamin C/E Hemoglobinopathies↑ erythrocyte pH
Erythrocyte destruction Splenectomy HemoglobinopathiesChronic renal failureSplenomegalyRheumatoid arthritisHAART meds,
Assays HyperbilirubinemiaCarbamylated HbETOHChronic opiates
Hypertriglyceridemia
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2. Insulin Levels
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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
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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
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3. Islet Cell Antibody (ICA)
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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)
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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
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4. C-peptide Test
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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
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Sweating
Palpitations
Hunger
Confusion
Blurred vision
Fainting
In severe cases, seizures
loss of consciousness
When is it ordered?
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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
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Not Only Test, But ….?
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