DAVAO DOCTORS COLLEGE

MEDICAL LABORATORY SCIENCE DEPARTMENT

STUDENT NOTES: CLINCHEM1
Prepared by: Merryl Louise C. Matus, RMT

PRELIM 1: ANALYTICAL TECHNIQUES AND Wavelength (nm) Color Complementary Color

INSTRUMENTATION 650-700 Red Green
600-650 Orange Blue
Basic Concepts and Terminologies 550-600 Yellow Indigo
 Electromagnetic Radiation-are described as photons of 500-550 Green Red
energy traveling in waves 450-500 Blue Orange
400-450 Indigo Yellow
350-400 Violet Yellow
 Complementary colors absorb each other.
 Each colored solution absorbs a unique pattern of
wavelengths
 In general, the perceived color of a solution will be
dominated by the wavelength transmitted or emitted by the
solution
 Crest or peak is the highest point while trough is the lowest Instumentation Techniques
point 1. Spectrophotometry
 Wavelength is the distance of two peaks/crest or troughs  Principle: Measures the amount of light transmitted to
when light travels in a wavelike manner determine the concentration of the light-absorbing substance
 Units: Angstrom (A)Millimicrons (mu) Nanometer (nm) in the solution; the measurement of the light transmitted by a
 Conversion factors: 1 nm = 10 A; 1 nm = 1 mu; 1 A = 10 mu
solution to determine the concentration of the light-absorbing
 Frequency is the number of waves that pass an observation
substance in the solution
point in a unit of time
 Beer-Lamberts Law states that the concentration of
 Amplitude is the distance between two adjacent peak and
substance is directly proportional to the amount of light
trough absorbed but inversely proportional to the logarithm of
transmitted light
%T = ratio of the radiant energy transmitted, divided by the
radiant energy incident on the sample
Absorbance (OD-optical density) = amount of light
absorbed
Components of the Spectrophotometer
 Wavelength is inversely proportional to the frequency of
the light wave
 Energy is inversely proportional to the wavelength of light
 The closer the two peaks are, the shorter the wavelength
 The shorter the wavelength, the larger number of photons will
be contained in a given distance
 More photons represents more energy, hence, shorter
wavelengths represent higher energy
 Light source-provides electromagnetic radiation as visible,
 Wavelength or frequency of the electromagnetic waves are
infrared, or UV light
perceived as color or hue
 Height or amplitude of the electromagnetic waves are a. Tungsten/Tungsten-iodide lamp– ideal for emission
perceived as intensity or brightness of light within the visible region (iodide prolongs
 There are two kinds of wavelengths: Visible Spectra which stability of Tungsten); produces energy wavelength
can be observed at 340-700nm and the Invisible Spectra from 340-700nm (visible region); used for moderately
which could either bw Ultraviolet with a wavelength of less diluted solution
than 400nm or Infrared with a wavelength of greater than b. Mercury Vapor Lamp – can emit UV light
700 nm. c. Deuterium Discharge Lamp - energy wavelength UV
 Other types of electromagnetic energy includes Cosmic range (down to 165nm)
Rays, Gamma Rays, X-rays and Appliances (Radio, TV, d. Infrared Energy Source- above 800 nm
Microwave, Etc.)
 e. Quart Halide Lamp- contains small amt of halogen
such as iodine to prevent the decomposition of
vaporized tungsten
f. Mercury Vapor Lamp- exists narrow bands of energy
at well defined places in the spectrum UV and visible
light
g. Hollow Cathode Lamp- consists of a gas-tight
chamber containing anode, a cylindrical cathode and
insert gas such as helium
 Entrance Slit- reduces stray light and prevents scattered
light from entering the monochromator
 Monochromator- isolates the specific wavelength of choice
a. Prism
-wedge-shaped pieces of glass, quartz, or sodium
chloride that allows transmission of light wherein each
side of the prism has different thickness allowing
selection of wavelength of light
-disperses white light into a continuous spectrum of
colors based on variation of refractive index for different
wavelength
b. Gratings
-has small grooves cut at such an angle that each
grooves behave like a very small prism and the
wavelengths are bent as they pass a sharp corner
-separate white light into various color comp.
c. Colored Filters
-made of glass that absorbs some portion of the
electromagentic spectrum and transmit others wherein
light energy is absorbed by dye components on the
class and is dissipated as heat
-band pass is 35-50nm or more
d. Interference Filters
-enhances desired wavelength by constructive
interference and eliminates others by destructive
interferences
-utlizes the wave cx of light to enhance the intensity of
the desired wavelength by constructive interference and
eliminates others by destructive interference and
reflections
-band pass is 10-20nm
 Exit Slit
 Analytical Cell- used to hold the solution in the instrument
whose concentration is to be measured
a. Borosilicate Glass- alkaline solution that do not etch
glass
b. Quartz or Plastic- best for wavelength below 320nm
c. Aluminum Silica Glass- best for visible light
d. Soft glass- best for acidic solution
 Detector- converts transmitted light energy into an equivalent
amount of electrical energy
a. Barrier layer cells- composed of film of light sensitive
material; no power source needed
b. Photoemission tube- has photosensitive material that
gives off electron when light energy strikes it; requires
an outside voltage for operation
c. Photomultiplier tube- used a series of electrodes to
internally amplify the photosignal before leaving the tube
 Meter (read out device)- simplest method of displaying output
of the detection system
2. Flame Emission Spectrophotometry
 Principle: measures the light emitted when electrons in an
atom become excited by heat energy produced by the flame
 Measures electrolytes with a 1+ charge: Na, K, Li
 Excited atoms return to ground state by emitting light energy
that is characteristic of that atom
 Analyte and color emitted:
Sodium filter- transmits only yellow light (589 nm)
Potassium filter- transmits only violet light (367 nm)
Lithium filter- transmits only red light (767nm)
Components of FES
 Flame
-breaks the chemical bonds to produce atoms
-source of energy that will be absorbed by the atoms to enter
the excitation state
 Gases
-using a mixture of hydrogen and oxygen gas (acetylene,
propane or natural gas)
 Atomizer or Burner
-breaks up the solution into finer droplets so that the atom will
absorb heat energy from the flame and get excited
a. Total Consumption Burner- aspirate sample directly into
flame
b. Premix Burner- involves the gravitational feeding of
solution
 Interference Filter- serves as monochromator
 Photocell- serves as photodetector
Internal Standard
Lithium is the preferred internal standard which also acts
as a radiation buffer
Criteria for Internal Standard
a. Concentration should be precisely the same in all
samples and standards
b. Energy must be close to the required amount to excite
the measured element
c. Normally found in solution being analyzed
3. Atomic Absorption Spectrophotometry
 Principle: measures concentration of the element by
detecting absorption of electromagnetic radiation by atoms
 The elements are not excited but are dissociated from their
chemical bonds and placed in the unionized, unexcited
ground state
 Measures electrolytes with a 2+ charge: Ca2+, Mg2+
Components of AAS
 Light Source- hollow cathode lamp, which produces a
wavelength of light that is specific for the kind of metal in the
cathode
 Mechanical Rotating Chopper- modulates light beam
coming from the light source
 Burner- uses flame to dissociate the chemical bonds and
form free unexcited atoms
a. Total Consumption Burner
ADV: flame is more concentrated and hotter
DISADV: produces large droplets in the flame; noisier
b. Premix Burner- gases are mixed; sample is atomized
before entering the flame
ADV: greater absorption and sensitivity; less noisy; large
droplets go to the waste
DISADV: flame is less hot
 Monochromator- selects the desired wavelength from a
spectrum of wavelength
 Detector
 Read-Out Device
 Interferences:
o Chemical – situation at which the flame could not
dissociate the sample into neutral atoms
o Ionization – situation at which atoms in the flame
become excited and emits energy
4. Volumetry/Titrimetry
 Principle: Unknown samples are made to react with a known
solution in the presence of an indicator
 Sample tests: Schales & Schales method;EDTA Titration
5. Gravimetry
 Principle: it is the isolation of the pure form of the sample
and its derivatives and the determination of its dry weight
 Sample tests: Lipid determination
6. Turbidimetry
 Principle: Measures the amount of light blocked by a
suspension of particular matter as light passes through the
cuvette
 Factors affecting measurement:
o Size and number of particles
o Depth of the tube
o Cross-sectional area of each particle
7. Nephelometry
 Principle: Measures the amount of light scattered by small
particles at an angle to the beam incident on the cuvette
 Factors affecting measurement same as turbidimetry
8. Flow Cytometry
 Principle: measures multiple properties of cells suspended
in a moving fluid medium
 Process:
a. All cells pass a single-file through a sensing point, where
they are intercepted by a laser beam
b. Cell suspensions are introduced into the flow chamber.
c. As the cells will pass through the flow chamber, they are
surrounded by low-pressure sheath that creates laminar
flow forcing the specimen into the center.
d. Each of the cells is intersected by light
e. The laser light excites the dye which emits a color of
light that is detected by the photomultiplier tube, or light
detector.
f. The cells are then sorted based from their electrical
charge
g. As the drop forms, an electrical charge is applied to the
stream to form a charge
h. This charged drop is then deflected left or right by
charged electrodes and into waiting sample tubes.
i. Drops that contain no cells are sent into the waste tube.
The end result is three tubes with pure subpopulations
of cells.
j. The number of cells is each tube is known and the level
of fluorescence is also recorded for each cell.
 Interpretation
Forward light scatter = cell size
90° angle scatter = cell granularity and nuclear irregularity
9. Chromatography
 Principle: involves the separation of a mixture on the basis
of specific differences of the physical and chemical
characteristics of the different components on a supporting
medium
 the constituents of the mixture are separated by a continuous
redistribution between two phases:
o mobile phase: carries the complex mixture
o stationary phase: where the mobile phase flows
 Types of Chromatorgraphy
a. Paper Chromatography
Principle: A spot of the substance fractioned is placed
on the paper just above the solvent level
The organic solvent moves up through the paper by
capillary action and variations in the sample move at
different rates
Basis of Separation:
o Rate of diffusion
o Solubility of solute
o Nature of the solvent
Clinical use: fraction of sugars, AA and barbituates
b. Thin Layer Chromatography
Same principle as paper chromatography but differs in
the sorbent used
SORBENT: thin plastic plates impregnated to a layer of
silica gel, alumina, polyacrylamide gel or starch gel
c. Liquid-Liquid Chromatography
Principle: separation of substances according to their
solubility in an organic/non-polar solvent and in an
aqueous/polar solvent
“Like Dissolves Like”
o highly polar substance = more soluble in a
highly polar solvent (water)
o lesser polar substance = more soluble in a
less polar solvent (organic substance)
Clinical use: fractionation of barbituates and lipids
 d. Ion Exchange Chromatography 13. Amperometry
Principle: the use of a resin (the stationary solid phase)  Principle: measures the amount of current that flows when
is used to covalently attach anions or cations onto it constant voltage is applied the measuring electrode
e. Column Chromatography
Principle: adsorption of the solutes of a solution through 14. Coulometry
a stationary phase and separates the mixture into  Principle: measures the amount of electricity (coulombs) at a
individual components fixed potential
Basis of Separation:  1 coloumb = 1 amper per second
 Faraday’s Law: number of coulombs consumed can be
o difference in pH
directly related to the concentration of the unknown
o polarity of solvent
Clinical use: Fractionation of sugars
15. Fluorimetry
f. Gel Chromatography
 Principle: Measures the fluorescence or the energy
Principle: the use of a resin (the stationary solid phase)
emission that occurs when a certain compound absorbs
is used to covalently attach anions or cations onto it
electromagnetic radiation, become excited and then return to
Basis of Separation:
an energy state that is usually higher than their original level
o Molecular weight & size
 Emitted light has longer wavelength than the incident/excited
o Charge of ions
light due to the loss of energy during collision
o Hydrophobicity of the molecules
 Phosphorescence- energy is equal to or lower than the
*Hydrophilic gels- soluble in aqueous medium absorbed energy (rarely happens)
ex. dextran, agarose, polyacrylamide  has 2 monochromators
*Hydrophobic gels-soluble in organic solvents  Main problem w/ fluorescence: Quenching
ex. methylatede sephadex, polystyrene beads
Clinical use: fraction of polysaccharides, NA, proteins, 16. Electrophoresis
enzymes, isoenzymes  Principle:migration or movement of charged particles in an
g. Gas Chromatography electric field
Principle: separating and measuring nanograms and  Factors affecting rate of migration:
pictogram amounts of volatile substance a. Net electric charge
Kinds of GC: higher electric charge = faster migration
o Gas-Solid Chromatography- sorbent is solid b. Size & Shape of Molecule
w/ a large surface bigger molecules = slower migration
o Gas-Liquid Chromatography-sorbent is a c. Electric Fluid Strength
non-volatile liquid higher ionic strength = slower movement
Basis of Separation: d. Temperature
o sample volatility more voltage = more movement
o rate of diffusion into liquid layer of the column *Problem with increased temp:
packing o Denaturation of Proteins
o solubility of sample in the liquid layer o Evaporation of solvent increases ionic strength
Clinical use: drug screening and drug analysis e. Nature of Supporting Media
fractionation of steroids, lipids, barbituates, blood o Paper Electrophoresis- earliest support media
alcohol and other toxicologic substances Disadv: paper is fragile and easily damaged staining of
protein
10. Potentiometry o Starch Gel Electrophoresis- good for large samp
 Principle: measures the difference in voltage at a constant Disadv: fragile and unable to store results permanently
current
o Cellulose Acetate Electrophoresis- strip with a clear
 Nerst Equation: relationship between the measured voltage
plastic backing w/ a coating of cellulose acetate
and the unknown concentration
particles attached to it
Disadv: becomes brittle when dried
11. Polarography
 Principle: measurement of difference in current at a o Agarose Electrophoresis
constant voltage Disadv: electric neutrality; separation is strictly on the
 Ilkovic Equation: relationship between the difference in basis of electric charge and uniformity of material size
current and voltage o Polyacrylamide Gel Electrophoresis- uses protein
size as the major factor in the separation process and
12. Conductometry the net charge of proteins
 Principle: measures the current flow between two non-  Specimens: Serum, Urine, Cerebrospinal Fluid
polarizable electrodes between a known electrical potential is  Clinical Use: analysis of proteins (serum) that can provide
established quick and useful information regarding the presence or
absence of disease entities
17. Automation
 Advantages:
o rapid results
o increases number of tests performed
o saves time and effort
o eliminates the need for more staff
o economical
o reduces errors in calculation and transcription
o better precision and accuracy
 Basic Approaches in Automation
a. CONTINUOUS FLOW ANALYZER
-Sequential analysis
-Uniformity in test performance
b. DISCRETE ANALYZER
-Separate analysis
-Most popular and versatile analyzer
c. CENTRIFUGAL ANALYZER
-Batch analysis
-Centrifugal force moves the reagents and sample to a
mixing chamber, into a cuvette, passing a light beam
and measuring the absorbance
PRELIM 2: QUALITY ASSURANCE AND QUALITY
CONTROL
 Quality Assurance overall program that ensures that the
final results reported by the laboratory are correct. It is
concerned with all factors that affect the test results from pre-
analytic, analytic and post-analytic phases.
 Quality Control is the system of ensuring accuracy and
precision in the lab and the process of ensuring that
analytical results are correct.
 QC samples are measured periodically in the same manner
as clinical samples
 The goal of Quality Control is to check the stability of the
machine. check the quality of the reagent and to check for
technical /clerical error
 Kinds of QC:
o Intralab- analyses of control samples together with patient
specimens; important in daily monitoring
o Interlab- involves proficiency testing programs that
periodically provide samples of unknown concentration of
analytes to participating laboratories (ex. NEQUAS);
maintains long-term accuracy of methods
 Characteristics of an Ideal QC Material:
o Resembles human sample
o Inexpensive and stable for long periods
o No communicable diseases
o No matrix effect
o With known analyte concentrations
o Convenient packaging for easy dispensing and storage
 Types of Reagents
o Reagent Blank- reagents without analyte added
o Standard- Most specific analytical solution; Value will
tell the concentration of the unknown; Only one analyte
o Control-Value will determine accuracy and precision;
Resembles patient sample; Many analytes
 Screening Test vs. Diagnostic Test
o Screening test
-done on apparently healthy individuals
-not a basis for treatment
-less accurate, less expensive
o Diagnostic Test
-Done on sick or ill individuals
-Basis for treatment
More accurate, more expensive
Quality Control Parameters
 Validity refers to how well the test measures what it is
supposed to measure. It refers to the accuracy of the test,
meaning how close the result is to its true value.
 Reliability refers to how well the test performs in use over
time. It refers to the precision of the test, meaning how close
are the results of a test on repetition or how close the values
are from one another.
 True positive (TP)—Indicating a person has the disease
when, in fact, he or she does.
 False positive (FP)—Indicating a person has the disease
when, in fact, he or she does not.
 False negative (FN)—Indicating a person does not have the
disease when, in fact, he or she does.
 True negative (TN)—Indicating a person does not have the
disease when, in fact, he or she does not.
 Sensitivity is the ability of test to detect the smallest amount
of analyte. Diagnostic Sensitivity refers to the ability of the
test to identify correctly those who have the disease (a) from
all individuals with the disease (a+c).
Formula: [TP / (TP + FN)]
 Specificity is the ability of test to detect substances without
interferences. Diagnostic Specificity refers to the ability of
the test to identify correctly those who do not have the
disease (d) from all individuals free from the disease (b+d).
Formula: [TN / (TN + FP)]
Statistics
 Mean- measure of central tendency
 Median- Middle value
 Mode- Most frequent value
 Range- Highest value – lowest value
 Standard deviation and variance are measures of
dispersion
 Coefficient of Variation is the index of precision
 Types of Errors
 Systematic errors are predictable errors; an error that is
constant when measurements are made under the same
conditions
Causes: deterioration of reagents, improperly made standard
solutions , contaminated solutions, calibration problems,
failing instrumentation
 Random errors are unpredictable errors; often due to
instrument, operator, and environmental conditions
Causes: pipetting error, mislabeling of samples, temperature
fluctuation, improper mixing of sample and reagent
Quality Control Charts: Levy-Jennings Chart
 The Levy-Jennings Chart is the most commonly used
histogram in QC
 A histogram is/are sheets of rectangular coordinate graphing
paper where data for sequential analysis are plotted to locate
the source of error.
 When results are In Control, results from QC fall within the
confidence limit.
 When results are Out of Control, the values of the control
fall outside the confidence limit.
 A Trend is formed by the control that continue to either
increase or decrease for a period of 6 or more consecutive
days
 A Shift is formed by the control that distribute themselves on
one side (above or below) of the mean, with no tendency
toward either a consistent fall or rise
 Outlier/s are values which are far from the main set of values
due to wild errors
Wesguard Rules
 The Wesguard Multirule System is a “multi-rule” system
developed by Dr. James O. Westgard based on statistical
concepts which is a combination of decision criteria or rules
to assess if a system is in control.
 o Galactose
12SD : It is violated if the IQC
value exceeds the mean by
2SD.
13SD : It is violated when the
IQC value exceeds the mean
by 3SD.
22SD : It detects systematic
errors and is violated when two
consecutive IQC values exceed
the mean on the same side of
the mean by 2SD.
R4SD : One control exceeding
the +2s and another exceeding
the -2s.
41SD : It is violated if four consecutive
IQC values exceed the same limit
(mean  1SD)
10x : This rule is violated when 10
consecutive IQC values are on the
same side of the mean or target
value
PRELIM 3: CARBOHYDRATES
 Carbohydrates are organic compounds composed of
carbon, hydrogen and oxygen
Chemical composition: Cn(H2O)n
 Two forms of CHO:
o Aldose- terminal carbonyl group called aldehyde group
o Ketose- carbonyl group in the middle linked to 2 other
carbon atoms
 Carbohydrates are the major constituents of physiologic
system: brain, erythrocyte, and retinal cells in humans.They
are also the major source of energy.
 Glucose is directly used as energy source and or stored as
glycogen in the liver or muscles.
Classification of CHO based on the number of
sugar units
 Monosaccharides
-simple sugars that cannot be hydrolyzed to a simpler form
- can contain 3 or more carbon atoms
o Glucose: most important CHO; major metabolic fuel
o Fructose
 Disaccharides
-two monosaccharides are joined by a glycosidic linkage
o Sucrose: Fructose + Glucose
o Lactose: Galactose + Glucose
o Maltose: Glucose + Glucose
 Polysaccharides
-linkage of many monosaccharide units
-on hydrolysis, will yield more than 10 monosaccharides
o Starch
o Glycogen: storage form of glucose in the body
o Chitin: fibrous substances consisting of polysaccharides
and forming the major constituent in the exoskeleton of
arthropods and the cell wall of fungi
Glucose Metabolism
 As food enters the mouth and oral cavity, food begins to be
broken down by ptyalin, an enzyme produced by the parotid
gland that helps in the initial metabolism of food
 When food reaches the stomach, the acidity inactivates
ptyalin and acid hydrolysis occurs. There is no carbohydrate
 digestion in the stomach, but protein digestion happens
through the enzyme pepsin.
 When food reaches the intestines, amylopsin, an enzyme
produced by the pancreas, further degrades the food and
convert polysaccharides into monosaccharides.
 Glucose then enters the bloodstream and increases glucose
uptake by the cells.
 Glucose metabolism
o After 30 min- glucose levels rise
o After 1 hour- gluose levels peak
o After 2 hours- glucose levels go back to normal
Glucose Metabolic Pathway
 Embden-Meyerhoff Pathway- provides energy for the body
o Aerobic – glucose to pyruvate
o Anaerobic – glucose to lactate
 Hexose- Monophosphate Shunt- production of reduced
NADPH and ribose-5-phosphate
 Glycogenesis
Processes in Regulating Glucose Metabolism
 Glycolysis – glucose to pyruvate or lactate to produce
energy
 Glycogenolysis – breakdown of glycogen to glucose for
energy
 Gluconeogenesis – formation of glucose-6-phosphate from
non-carbohydrate sources
 Glycogenesis – glucose to glycogen for storage
 Lipogenesis – conversion of carbohydrates to fatty acids
 Lipolysis – Decomposition of fat
Hormones Regulating Glucose Metabolism
 Insulin
- produced by the β cells of the islets of Langerhans in the
pancreas
- only hormone that decreases glucose
- promotes glycolysis, glycogenesis, lipogenesis
- Proinsulin: immediate precursor of insulin
- C-Peptide: test that is based on the presence of proinsulin
that helps in the differential diagnosis of Type 1 from type 2
Diabetes Mellitus and the diagnosis of insulinomas
 Glucagon
- produced by the α cells of the islets of Langerhans in the
pancreas
- primary hormone that decreases glucose for increasing
glucose levels
- promotes glycogenolysis and gluconeogenesis
 Somatostatin
- produced by the δ cells of the islets of Langerhans in the
pancreas
- inhibition of pancreatic hormone release of insulin and
glucagon
- inhibition of gastric acid secretion
 Cortisol
- produced by the zona fasciculata of the adrenal cortex
- promotes hepatic gluconeogenesis and lipolysis
 Catecholamines
- produced by the chromaffin cells of the adrenal medulla
- inhibits insulin secretion and promotes glycogenolysis
 Thyroid hormones
- produced by the thyroid gland
- promotes glycogenolysis and intestinal absorption of
glucose
 Growth Hormone
- produced by the anterior pituitary gland
- promotes glycogenolysis and lipolysis
 Adenocorticotropic Hormone
- produced by the anterior pituitary gland
- promotes glycogenolysis and gluconeogenesis
Hypoglycemia
 Hypoglycemia- low blood glucose levels
 May be due to insulinoma or diabetic shock
 Reference Values
o 65-70 mg/dL = glucagon and other glycemic factors start to
increase
o 55 mg/dL = observable symptoms; strongly suggest
hypoglycemia
o <40 mg/dL = panic value
o 20-30 mg/dL = severe CNS dysfunction
 Whipple’s Triad
1. Symptoms present
2. Low blood glucose
3. Relief of symptoms when glucose is raised to normal
Hyperglycemia
 Hyperglycemia- high blood glucose levels
(FBS >126mg/dL)
 Causes: stress, severe infection, dehydration or pregnancy,
pancreatectomy, insulin deficiency or abnormal insulin
receptor
 Laboratory findings
o increase glucose in plasma and urine
o increase urine specific gravity
o ketones in serum and urine
o decrease blood and urine pH
o electrolyte imbalance
 Values greater than 500 mg/dL may result to multiple organ
failure (nephropathy, neuropathy, retinopathy)
Diabetes Mellitus
 Diabetes Mellitus is the most common type of
hyperglycemia that can either be due to insufficient/complete
absence level of insulin or insulin resistance
 Clinical signs and symptoms include:
o Polyuria- excessive urination
o Polydipsia- excessive thirst
o Polyphagia- increased food intake
 Glucosuria is the presence of glucose in the urine
 Microalbuminuria is the slight increase of albumin in the
urine that is due to the kidneys leaking out small amounts of
albumin passed through the urine
 Diagnostic Criteria
o increased Signs and symptoms of DM
o FBS = ≥ 126 mg/dL or ≥ 7.0 mmol/L
o Non-diabetic < 100 mg/dL
 o Pre-diabetes 100-125 mg/dL
o RBS = ≥ 200mg/dL or ≥ 11.1 mmol/L
o OGTT = ≥ 200mg/dL or ≥ 11.1 mmol/L
o HbA1c ≥ 6.5%
o decrease blood and urine pH
Type I Diabetes Mellitus
 also known as Insulin dependent DM, Brittle diabetes,
Juvenile onset DM
 caused by the cellular-mediated autoimmune destruction of
the beta cells of the islets of Langerhans in the pancreas
 signs and symptoms: Rapid weight loss, hyperventilation,
mental confusion, possible loss of consciousness
 Prone to ketoacidosis
 C-peptide: Undetectable to very low
 Treatment or management include Exogenous Insulin
Therapy
Type II Diabetes Mellitus
 also known as Non-insulin dependent DM, Stable
diabetes, Adult onset DM
 caused by insulin receptor defect leading to insulin resistance
 signs and symptoms: Weight gain, milder symptoms than
Type I DM
 Not prone to ketoacidosis
 C-peptide: detectable/high
 Management: Healthy lifestyle
Gestational Diabetes Mellitus
 pregnancy onset usually in the second trimester due to
hormonal imbalance
 Occurs during pregnancy and should disappear after delivery
 May lead to Type 2 DM after 5-10 years if not treated
 Infants born to diabetic mothers are at increased risk for
respiratory distress syndrome, hypocalcemia, and
hyperbiliruninemia
 Screening should be performed between 24th and 28th
weeks of gestation (1-hr Glucose Challenge Test using 50g
glucose load)
Specimen Considerations
 Whole blood, serum, plasma, CSF, urine, synovial fluid
can be used as specimens for glucose testing
 Requires atleast 8-10 hours of fasting
 Glucose is metabolized at a rate of:
7 mg/dL /hr at Room Temp 2 mg/dL /hr at 4°C
 Whole blood glucose levels are 10-15% lower than serum
blood glucose levels
Point of Care Testing for Glucose
 Using only a drop of capillary blood, the reaction on the
reagent pad is read by reflectance colorimeter
 Glucose value of >140 mg/dL should be rescreened with
FBS, HbA1c, or OGTT using venous samples.
 should not be used to diagnose diabetes or other
hyperglycemic conditions
Glucose Testing
 Fasting Blood Sugar (FBS)
- screening test for DM
- detect elevation of blood glucose
- 8-10 hours fasting is required; not more than 16hrs
- produced by the β cells of the islets of Langerhans in the
pancreas
Categories of fasting plasma glucose
- Normal = <110 mg/dL
- Impaired fasting glucose = >110- <126 mg/dL
- Provisional Diabetes dx = >126 mg/dL
 Random Blood Sugar (RBS)
- sugar determination of randomly collected blood
- requested during insulin shock or hyperglycemic ketonic
coma
 2-hr Postprandial Blood Sugar
- FBS is first performed
- Patient is then given a glucose load (75g)
- plasma glucose is determined after 2hrs
- NORMAL: blood glucose will return within reference limits
after 2 hours
 Oral Glucose Tolerance Test (OGTT)
- series of glucose testing
- FBS is first performed
- Patient is then given a glucose load
- collect blood specimen after 30 min, 1 hr and 2 hrs after
glucose load intake
Guidelines
- consume a normal to high carbs intake (150g of carbs per
day for 3 consecutive days prior to the test)
- discontinue medication that affects glucose levels
- fast overnight; should be performed in the morning
- Px should be ambulatory; avoid exercise, eating/drinking
and smoking
- FBS is measured before giving the glucose load
*if FBS >140 mg/dL = stop test; if < 140 mg/dL, proceed
- glucose load: 75g for adult; 100g for pregnant; 1.75g/kg wt
for children
- drink glucose load within 5-15 min without puking
 Intravenous Tolerance Test (IVTT)
- for those who cannot take large carbohyrate intake, altered
gastric physiology, undergone operation in intestine,
chronic malabsorption syndrome
 Insulin Tolerance Test (ITT)
- evaluates sensitivity to insulin
- glucose is administered orally 30min after insulin
 Other tolerance tests
Galactose Tolerate Test
- detects impaired glycogenolysis or severe liver damage
Leucine Tolerance Test
- used for children suffering from hypoglycemic episodes
Tolbutamide Tolerance Test
- to evaluate the hypoglycemic effects by insulinomas
 Glycosylated haemoglobin (HbA1c)
- Hba1c is formed by attachment of glucose to the beta
chains of Hemoglobin A1
- Long term monitoring of glucose
- Detects glucose levels in the average of 2-3 months since
the RBC lifespan in the circulation is 120 days
- Not suitable for patients with RBC lifespan disorders
 - Specimen: Whole blood EDTA  Dubowski Method (o-Toluidine Method)
- Normal value: < 6.5% glucose + 0-toluidine + acetic acid + heat = green complex
- (5.7-6.4% = prediabetes) (630 nm)
 Fructosamine - most specific non-enzymatic method for glucose
- Fructosamine is formed by attachment of glucose to
albumin Enzymatic Methods
- Short term monitoring of glucose  Glucose oxidase method (oldest method)
- Detects glucose levels in the average of 2-3 weeks since - Principle: glucose exists as α (35%) or β (65%)
the lifespan of albumin in the circulation is 20 days mutarotase converts alpha to beta glucose
- Not suitable for patients with hypoalbuminemia - False results: increased ascorbate, bilirubin, uric acid,
- Specimen: Serum glutathione, creatinine, l-cysteine, l-dopa, dopamine,
- Normal value: 205-285 µmol/L methyldopa, and citric acid
 Saifer-Gernstenfield Method (Trinder Reaction)
Methods for Glucose Testing glucose + O2  gluconic acid + H2O2
Copper Reduction Methods (oldest method) H2O2 + chromogenic subs  H2O + oxidized chromogen
- Principle: glucose in a hot alkaline solution readily coupled enzymatic method ( GO and Peroxidase)
reduces cupric ions to cuprous ions  Hexokinase method (highly specific and reference method)
 Folin Wu Method - False results: hemolyzed and icteric spx
Cuprous + phosphomolybdate = phosphomolybdenum blue Glucose + ATP  Glucose-6-phosphate + ADP
- Disadv: non-glucose reducing sugars are not precipitated G-6-P + NADP  6-phosphogluconolactone + NADPH
during process coupled enzymatic method (G6PD and Hexokinase)
 Nelson Somogyi Method
Cuprous + arsenomolybdate = arsenomolybdenum blue Inborn Errors of Carbohydrate Metabolism
- Adv: non-glucose reducing sugars are adsorbed by barium  GLYCOGENOSES = defect in glycogen metabolism
sulfate  consequence of inherited deficiencies of enzymes that
 Neocuproine Method control the synthesis or breakdown of glycogen
Cuprous + Neocuproine reagent = yellow-orange complex  Can either cause abnormal quality or quantity of glycogen
- Reagent: Neocuproine rgt (2,9-dimethyl-1,10-  Liver (hypoglycemia and hepatomegaly) and muscles
phenanthroline HCl (muscle cramps, exercise intolerance, fatigue, weakness) are
 Shaffer-Hartman Somogyi Method commonly affected
Cuprous + Iodine + Acidic solution = colorless complex
- excess iodine is titrated with thiosulfate TYPE DEFECT
 Benedict’s Method (modified Folin Wu method) Type I Von Gierke disease Glucose-6-phosphatase
Copper sulfate (blue) + glucose + heat = brick red Type II Pompe disease Lysosomal acid α-
glucosidase
precipitate
Type III Cori or Forbe’s Glycogen debranching
disease enzyme
Alkaline Ferric Reduction Method
Type IV Anderson’s disease Glycogen branching
 Hagedorn-Jensen Method enzyme
Ferricyanide (yellow) + glucose = ferrocyanide (colorless) Type V McArdle disease Muscle phosphorylase
- inverse colorimetry; measured at 420 nm Type VI Hers disease Glycogen phosphorylase
(liver)
Condensation Method Type VII Tarui disease Phosphofructokinase
 Condensation with Phenols Type IX Phosphorylase kinase
aldehyde group + phenol = colored complex (liver)
- Common reagents: anthrone, thymol, phloroglucinol, Type XI Fanconi-Bickel GLUT 2
resorcinol, alpha naphthol, m-aminophenol disease
 Condensation with Aromatic Amine Type 0 Glycogen Synthase
- Common reagents: o-toluidine, aniline, 2-aminophenyl