diabetic profile - lab diagnosis

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


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.


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

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


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 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


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 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


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

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


Calculation

Glucose conc. = Abs. Sample X Conc. Standard

Abs. Standard ..


CBC – Capillary Blood Glucose


Special Tests


1. GlycohaemoglobinHb 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 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


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


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


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

Health & Medicine

glucose oxidase

glucose concentration

plasma glucose

normal blood glucose

low renal threshold

diabetes mellitus tidmtype

blood liver

high blood sugar levels