Diabetic Profile

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Diabetic Profile
Ravi Kumudesh MSc/BSc/Dip(MLT)
Sri Lanka Society for Medical Laboratory
Science slsmls.org / kumudeshr_at_gmail.com
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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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Clinical Biochemical Findings in Diabetes

• 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
  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.

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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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Diabetic profile

• 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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1. Urine Sugar
Detection of urinary glucose (Glucosuria)
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Glucosuria

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

Positive Benedicts test
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• 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.
• Diastix can be directly applied to the urinary
  stream, and differing color responses of the
  indicator strip reflect glucose concentration.
• Benedicts and Fehlings test can also detect
  glucosuria.

Diastix- Reagent strips
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Microalbuminuria
2. Urine Microalbumin
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Microalbuminuria

• The importance of micro- albuminuria in the
  diabetic patient is that it is a signal of early
  reversible renal damage.
• Performing an albumin-to-creatinine ratio is
  probably easiest.
• 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

• May be defined as an albumin excretion rate
  intermediate between normality (2.5-25 mg/day)
  and macroalbuminuria (250mg/day).
• 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.

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Assays for Microalbuminurea
Qualitative

• Dipstick method

Quantitative

• 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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3. Urine Ketone Bodies
Ketonuria
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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 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

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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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How to Test Urine Ketones

• 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

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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

• Touch the blood drop to the purple area on the
  top of the test strip. The blood is drawn into
  the test strip
• Continue to touch the blood drop to the purple
  area on the top of the test strip until the
  monitor begins the test
• The blood ß-Ketone result shows on the display
  window with the word KETONE

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Ketonuria

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

Ketostix- Reagent strips
Positive Rotheras test
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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 dont 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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Procedure

• 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.

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Other types of OGTT

• 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.

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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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Interpretation

• Renal Glycosuria
• Curve is normal due to lowered renal threshold
  one or more samples of urine contain glucose.
• Lag storage/Alimentary Type
• FBS is normal. Due to rapid absorption, maximum
  level is found at 30 min (180mg/dl). glucosuria
  is seen hypoglycaemic levels may be reached at
  end of 2 hours. 
• Flat curve of enhanced glucose tolerance
• FBS is normal. Throughout the test the level
  does not vary 20mg.

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Hypoglycemia

• When blood glucose falls below 60 mg/dl.
• Causes
• Most commonly seen in overdose of insulin in
  treatment of DM.
• Hypothroidism.
• Insulin secreting tumours of pancrease rare.
• Hypoadrenahsm (Addison's disease)
• Hypopitruitism.
• Severe exercise.
• Starvation.

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Measuring glucose level

• Principle

Glucose H2O O2 Gluconic
acid H2O2 2H2O2 4 aminoantipyrine PHBS
Quinoneimine dye H2O


Red color
GOD
POD
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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

• Prepare the reaction as the following
• Mix, incubates at 37oC for 10min
• Read abs at 510nm

Sample Standard Reagent blank
1ml 1 ml 1 ml Glucose oxidase reagent
10 µl - Sample (serum)
- 10 µl - Glucose standard
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Calculation
Glucose conc. Abs. Sample X Conc.
Standard Abs. Standard ..
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Bedside Method
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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 A1c4.5-5.7
• Total glycohemoglobin5.3-7.5

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Key Messages

• Glycated hemoglobin (A1C) ? measure every 3
  months (6 months if stable at target)
• Self monitoring Blood Glucose (SMBG) is an aid to
  assess interventions and hypoglycemia
• Individualize the frequency of SMBG
• 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 glycemic targets are
  not being met or treatments adjusted
• Measure every 6 months if stable at glycemic
  targets

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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 B12 Reticulocytosis Chronic 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 Hemoglobinopathies Chronic renal failure Splenomegaly Rheumatoid arthritis HAART meds,
Assays Hyperbilirubinemia Carbamylated Hb ETOH Chronic opiates Hypertriglyceridemia
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2. Insulin Levels
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Insulin Test - Clinical Relevance

• 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.

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Insulin - Test Principle

• 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.

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3. Islet Cell Antibody (ICA)
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Demonstration of 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.

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Test Principle and Procedure

• 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

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4. C-peptide Test
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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

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When is it ordered?

• Sweating
• Palpitations
• Hunger
• Confusion
• Blurred vision
• Fainting
• In severe cases, seizures
• loss of consciousness

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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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Thank You !