Analytical Techniques &

Instrumentation

Light & Energy Principles

Electromagnetic Radiation
- photons of energy traveling in waves or
wavelike manner
- Radiant energy from short wavelength
gamma rays to long wavelength radio waves
- The shorter the wavelength, the higher the
2. Frequency (WF)
electromagnetic energy
- numbers of waves that pass an observation
point in a unit of time
Types of Electromagnetic Energy
1. Cosmic Rays
2. Gamma Rays
3. X-rays
4. Visible Rays
5. UV Rays
6. Infrared
7. Appliances (Radio, TV, Microwave, etc.)
3. Amplitude
- distance between two adjacent peak and
trough

THINGS TO REMEMBER
• Wavelength is inversely proportional to the
frequency of the light wave
• Energy is inversely proportional to the
wavelength of the light
• The closer the two peaks are, the shorter the
wavelength
• more photons = more energy
• shorter wavelengths = higher energy
PARTS OF A WAVE
4. COLOR/HUE AND BRIGHTNESS
- Color is dependent on the wavelength (WL)
o short WL = high WF (bluish)
o long WL = low WF (reddish)

1. Wavelength
- Distance of two peaks/crest or troughs when
light travels in a wavelike manner
- The distance between two identical parts in
consecutive waves.

UNITS:
Angstrm (A) 1nm = 0.1 A - Height/Amplitude is equal to
Millimicrons (mu) 1 nm = 1 mu intensity/brightness
Nanometer (nm) 1A = 0.1 mu o great amplitude = bright color; loud
sound
o small amplitude = dull color; soft
sound

Annie June Audan

 Analytical Techniques and Instrumentation

Kinds of Colorimetry
1. Visual Colorimetry
2. Photoelectric Colorimetry
a. Spectrophotometry
b. Filter Photometry
Primary Consideration
1. Quality of the Color
2. Intensity of the Color

INSTRUMENTATION TECHNIQUES

SPECTROPHOTOMETRY
Principle:
Measures the amount of light transmitted to determine
the concentration of the light-absorbing substance in
the solution; the measurement of the light transmitted
by a solution to determine the concentration of the
Kinds Of Wavelength
light-absorbing substance in the solution
1. Visible Spectra (340 – 700nm); 400 nm
BEER – LAMBERTS LAW
2. Invisible Spectra
- states that the concentration of substance is
o Ultraviolet = below 340 nm (<400
directly proportional to the amount of light
nm)
absorbed but inversely proportional to the
o Infrared = above 700 nm (>700nm)
logarithm of transmitted light
- Directly proportional to light absorb (amount)
COMPLEMENTARY COLORS
- Inversely proportional to the transmitted light
- Complementary colors absorb each other
(algorithm)
- Each colored solution absorbs a unique
%T = ratio of the radiant energy transmitted, divided
pattern of wavelengths
by the radiant energy incident on the sample
- The perceived color of a solution will be
Absorbance (OD-optical density) = amount of light
dominated by the wavelength transmitted
absorbed

Components of a Spectrometer

1. Light Source
- provides electromagnetic radiation as visible,
infrared, or UV light
Wavelength Complementary Examples:
Color
(nm) Color ✓ Tungsten/ Tungsten-Iodide Lamp – ideal for
650-700 Red Green emission of light within the visible region
600-650 Orange Blue (iodide prolongs stability of Tungsten);
550-660 Yellow Indigo produces energy wavelength from 340-
500-550 Green Red 700nm (visible region); used for moderately
450-500 Blue Orange diluted solution
400-450 Indigo Yellow ✓ Mercury Vapor Lamp – UV region
✓ Deuterium Discharge Lamp – energy
350-400 Violet Yellow
wavelength UV range (down to 165nm)
✓ Infrared Energy Source – IR region (above
800nm)
✓ Quart Halide Lamp – contains small amt of
halogen such as iodine to prevent the
decomposition of vaporized tungsten
✓ Mercury Vapor Lamp – exists narrow bands
of energy at well defined places in the
spectrum UV and visible light
✓ Hollow Cathode Lamp – consists of a gas-
tight chamber containing anode, a cylindrical
cathode and insert gas such as helium
2. Entrance Slit
- reduces stray light and prevents scattered
light from entering the monochromator

Annie June Audan

 3. Monochromator
- Isolates the specific WV of choice (single WL)
Examples:
✓ 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
✓ Diffraction 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 components
✓ Colored Filters
- Made of glass that absorbs some
portion of the electromagnetic
spectrum and transmits others
- Light energy is absorb by dye
components on the class and is
dissipated as heat
- Band pass is 25-50nm or more
✓ Interference Filters
- Enhances desired wavelength by
constructive interference and
eliminates others by destructive
interferences
- utilizes the wave character 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
Advantages of Gratings over Prisms
a. It produces linear spectrum and therefore
maintaining a constant band pass which is
simple.
b. It can be used in the regions of spectrum
where light energy is absorbed by glass prism.
4. Exit Slit
- Allows the transmission of a specific WL of
choice from monochromator to the sample in
cuvette
5. Cuvette (Analytical Cell/Sample Cup)
- Used to hold the solution in the instrument
whose concentration is to be measured
Examples:
✓ Borosilicate glass – alkaline sol’n that do not
etch glass
✓ Quartz or Plastic – best for WL below 320 nm
as it does not absorb UV radiation below 320
nm
✓ Aluminum Silica Glass – best for visible light
(340 nm and above)
✓ Soft Glass – best for acidic sol’n
✓ Plastic Cells – both visible and UV range
6. Detector
- Electron tube amplifying a current that can
convert transmitted light energy into an
equivalent amount of electrical or
photoelectric energy
Examples:
✓ Barrier Layer Cells – composed of film of light
sensitive materials like selenium on iron plate
with transparent layer of silver; no power
source needed
✓ Photoemission tube (Phototube) – has
photosensitive material that gives off electron
when light energy strikes it; requires an
outside voltage for operation; consists of 2
electrodes sealed in an evacuated glass to
prevent scattering of photoelectrons by
collision with gas molecules
✓ Photomultiplier tube – used a series of
electrodes to internally amplify the
photosignal before leaving the tube
7. Meter (read out device)
- Simplest method of displaying output of the
detection system
FLAME EMISSION SPECTROPHOTOMETRY (FES)
Principle: measures the light emitted when electron in
an atom become excited by heat energy produced by
the flame
• Also known as FILTER PHOTOMETRY/
EMISSION FLAME PHOTOMETRY
• 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 (G1 metal)
1. Light Source
2. Acetylene Flame Source
o Gas – using a mixture of hydrogen
and oxygen gas (acetylene, propane
or natural gas)
o Flame – breaks the chemical bonds to
produce atoms; source of energy that
will be absorbed by the atoms to
enter the excitation state
3. Atomizer/ Burner
- Breaks up the soln into finer droplets so that
the atom will absorb heat energy from the
flame and get excited
o Total Consumption Burner – aspirate
sample directly into flame, the gases
are passed at high velocity over the
end of the capillary suspended in the
soln.
Annie June Audan
 o Premix Burner – involves the
gravitational feeding of soln through
a restricting capillary into an area of
high velocity gas flow where small
droplets are produced and passed
into the flame.
4. Monochromator
- Uses interference filter
Analyte and color emitted
o Na (sodium) filter = yellow light (589
nm)
o K (potassium) filter = violet light (767
nm)
o Li (lithium) filter = red light (761 nm)
o Mg (magnesium) filter = blue light
5. Detector
- Uses photocell
THINGS TO REMEMBER
• Lithium preferred internal standard w/c also
acts as a radiation buffer (Na, K)
• Lithium emission is similar to Na and K
• It is normally present as trace elements and
has no interferences
• Use CESIUM if you are going to test for lithium
Criteria for Internal Standard
✓ Concentration should be precisely the same in
all samples and standards
✓ Energy must be close to the required amount
to excite the measured element
✓ Normally found in solution being analyzed
ATOMIC ABSORPTION SPECTROMETRY (AAS)
Principle: measures concentration of the element by
detecting absorption of electromagnetic radiation by
atoms, rather than by molecules.
• The elements are not excited but are
dissociated from their chemical bonds and
placed in the unionized, unexcited ground
state (G2 metal; Calcium, Magnesium)
• Measures electrolytes with a 2+ charge: Ca2+
& Mg2+
• Interferences:
o Chemical – situation at w/c the flame
could not dissociate the sample into
neutral atoms
o Ionization – situation at w/c atoms in
the flame become excited and emits
energy instead of staying in the
ground state
o Matrix – formation of solids from
sample droplets due to enhancement
of light absorption by organic
solvents
• More sensitive than flame emission
spectrometry
1. Light source
- Uses hollow cathode lamp which produces a
wavelength of light that is specific for the kind
of metal in the cathode
2. Mechanical Rotating Chopper
– modulates light beam coming from the light
source
3. Burner
- Uses flame to dissociate the chemical bonds
and form free unexcited atoms
✓ Total Consumption Burner
o ADV: flame is more concentrated and
hotter, lessening chemical
interferences
o DISADV: produces large droplets of
flame; noiser
✓ Premix Burner
– gases are mixed; sample is atomized before
entering the flame
o ADV: greater absorption and
sensitivity; less noisy; large droplets
go to the waste
o DISADV: flame is less hot and cannot
dissociate metal complexes
4. Monochromator
- selects the desired wavelength from a
spectrum of wavelength
5. Detector
- Uses photomultiplier tubes to measure the
intensity of the light signal
6. Read Out Device (meter)
VOLUMETRY/TITRIMETRY
Principle: unknown samples are made to react with a
known solution in the presence of an indicator
Sample test:
✓ Schales & Schales method (Chloride
determination)
✓ EDTA Titration (Calcium determination)
GRAVIMETRY
Principles: isolation of the pure form of the sample and
its derivatives and the determination of its dry weight
Sample Test:
✓ Total Lipid Determination
TURBIDIMETRY
Principles: measures the amount of light blocked by the
suspension of particular matter as light passes through
the cuvette
FACTORS AFFECTING MEASUREMENT
✓ Size and number of particles
✓ Depth of the tube
✓ Cross-sectional area of each particle
Disadvantage: variable absorption due to aggregation
of particles w/c tend to settle out of the soln.
Annie June Audan
 NEPHELOMETRY
Principle: measures the amount of light scattered by
small particles at an angle to the beam incident on the
cuvette
- More specific than turbidimetry
FLOW CYTOMETRY (FC)
Principles: measures multiple properties of cells
suspended in a moving fluid medium
• Core component of hematologic cell counters
Process:
1. All cells pass a single-file through a sensing
point, where they are intercepted by a laser
beam
2. Cell suspensions are introduced into the flow
chamber
3. As the cell will pass through the flow
chamber, they are surrounded by low-
pressure sheath that creates laminar flow
forcing the specimen into the center
4. Each of the cells is intersected by light
5. The laser light excites the dye which emits a
color of light that is detected by the
photomultiplier tube, or light detector
6. The cells are then sorted based from their
electrical charge
7. As the drop forms, an electrical charge is
applied to the stream to form a charge
8. This charged drop is then deflected left or
right by charged electrodes and into waiting
sample tubes
9. Drops that contain no cells are sent into the
waste tube. The end result is three tubes with
pure subpopulations of cells.
10. The number of cells in 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
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 called ABSORBENT or SORBENT
- the constituents of the mixture are separated
by a continuous redistribution between two
phases:
1. MOBILE PHASE
- Known as Eluent or Carrier Fluid
- Carries the complex mixture
- The constituents of the mixture are separated
by a continuous redistribution between two
phases
2. STATIONARY PHASE
- Where the mobile phase flows
Types of Chromatography
1. PAPER CHROMATOGRAPHY (PC)
- A spot of the substance fractioned is placed
on the paper just above the solvent level
The organic solvent (mobile phase) moves up through
the paper by capillary action and variations in the
sample move at different rates
Basis of Separation
✓ Rate of diffusion
✓ Solubility of solute
✓ Nature of the solvent
Clinical Use: fractions of sugar, amino acids, and
barbituates
2. THIN LAYER CHROMATOGRAPHY (TLC)
- Same principle as PC but different on the
sorbent used
- Most commonly encountered
Sorbent: thin plastic plates impregnated to a layer of
silica gel, alumina, polyacrylamide gel, or starch gel
3. PARTITION CHROMATOGRAPHY (liquid-
liquid) (LL)
- Separation of substances according to their
solubility in an organic/nonpolar solvent and in
an aqueous/polar solvent
Basis of Separation
✓ Differences in solubility between two liquid
phases
LIKE DISSOLVES LIKE
• Highly polar substances = more soluble in a
highly polar solvent (water)
• Lesser polar substances = more soluble in a
less polar solvent (organic substances)
Annie June Audan
Clinical Use: fractionation of barbituates and lipid
studies
4. GEL CHROMATOGRAPHY
- When a mixture of small and large molecules
allowed to pass over small particles in column,
the small particles diffuse into the gel,
whereas larger molecules tend to pass rapidly
in the column and appear in the eluate first.
- Other names: Gel Permeation, Size exclusion,
Gel Filtration, Molecular sieve/exclusion
Basis of Separation
✓ Molecular weight and size
✓ Charge of ions
✓ Hydrophobicity
1. Hydrophilic gels
- Soluble in aqueous solution
Examples: dextran, agarose, polyacrylamide
2. Hydrophobic gels
- Soluble in organic solvents (triglycerides &
fatty acids)
Examples: methylated sephadex, polystyrene
beads
Clinical Use: fraction of polysaccharides, Nucleic acids,
protein, enzymes, isoenzymes
5. ION EXCHANGE CHROMATOGRAPHY (IEC)
- The use of a resin (the stationary solid phase)
is used to covalently attach anions or cations
onto it.
o Anion = negative (-) charge
o Anode = positive
o Cation = positive (+) charge
o Cathode = negative
6. COLUMN CHROMATOGRAPHY (CC)
- Adsorption of the solutes of a solution through
a stationary phase and separates the mixture
into individual components
Basis of Separation
✓ Difference in pH
✓ Polarity of solvent
Clinical Use: fractionation of sugars
7. GAS CHROMATOGRAPHY
- Separating and measuring nanograms and
pictogram amounts of volatile (easy to
evaporate; escape as gas) substances
Types of Gas Chromatography
1. Gas Solid Chromatography
- Sorbent is solid with a large surface
2. Gas Liquid Chromatography
- Sorbent is a non-volatile liquid
Basis of Separation
✓ Sample volatility
✓ Rate of diffusion into liquid layer of the column
packing
✓ Solubility of sample in the liquid layer
Clinical Use: drug screening and drug analysis;
fractionation of steroids, lipids, barbituates, blood
alcohol, and other toxicologic substances
8. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
Principle: It follows the concept of selective absorption.
It applies 4,000 – 10,000 lbs/square inch pressure from
the rapid identification and separation of high
molecular weight components and man liable biologic
compounds (peptides, drugs, hormones, barbituates,
lipids, steroids & antibiotics)
FLUORIMETRY
Principle: measures the fluorescence or the energy
emission that occurs when a certain compound
absorbs electromagnetic radiation, become excited
and then turn to an energy state that is usually higher
than their original level.
- Since the energy of light given off is less than
that of absorbed, the WL of light given off is
longer than that absorbed for excitation.
• FLUORESCENT – the energy it gives up is
longer than it absorbs
• FLUOROPHORE – atom/molecule that
fluorescent
Annie June Audan
 • Phosphorescence - energy is equal to or lower ELECTROCHEMISTRY
than the absorbed energy (rarely happens)
• Emitted light has longer wavelength than the POTENTIOMETRY
incident/excited light due to the loss of energy Principle: Measures the difference in voltage at a
during collision constant current. The measurement of potential or
• Light source usually used is Mercury Vapor/ Xe voltage between 2 electrodes in soln by a null-balance
lamp that emits high intensity light technique.
• Uses 2 monochromators: - The unknown voltage introduced into the
o Excitation monochromator – receives potentiometer circuit opposes a known
light and emits light to cuvette reference voltage.
o Emission (secondary) monochromator - Uses the NERST EQUATION
– emits longer wavelength (= shorter o Relationship between the measured
energy); positioned at a RIGHT voltage and the unknown
ANGLE from cuvette to eliminate concentration
interference from excitation light
• QUENCHING – main problem with fluorescent;
the quick disappearance of fluorescent
Fluorescence concentration measurement is related to:
1. Molar absorptivity of the compound
2. Intensity of incident radiation
3. Quantum efficiency of every emitted per
quantum absorbed
4. Length of light path

SCINTILLATION COUNTER
Principle:
It is use to measure the disintegration per minute of
time of a radioisotope.

Types of Radiation:
1. Alpha – positively charged particles; POLAROGRAPHY
resembles the nucleus of a helium atom with Principle: Measurement of difference in current at a
mass of 4; have a very little of energy. constant voltage.
2. Beta – resembles an electron with both beta+ - It is used to measure trace metals (oxygen,
and beta- charge, but essentially no mass; vitamin C, amino acids concentration)
exists in forms: soft & hard beta. - Uses the ILKOVIC EQUATION
3. Gamma – a form of electromagnetic energy o Relationship between the difference
with no mass, only energy; exists in forms: soft in current and voltage
& hard gamma
Types of Scintillation Counters:
1. Solid Scintillation Counter
- measures gamma radiation using thallium
- activated NaL crystals as scintillator and PM
tube as detector with preamplifier circuit
2. Liquid Scintillation Counter
- Measures beta radiation using liquid flour as
scintillator

RADIOIMMUNOASSAY (RIA)
Principle: An immunologic procedure involving the use
of radioisotope.
Substances involved:
1. Unlabeled antigen – substances being AMPEROMETRY
analyzed Principle: Measures the amount of current that flows
2. Radiolabeled antigen – acts as label when constant voltage is applied the measuring
3. Antibody – provide binding site for the two electrode.
antigens - Equivalent point is established by changed in
Types of RIA average of current passing through a soln
1. Solid RIA during progress of titration of the said current
2. Liquid RIA being under an applied constant voltage.

CONDUCTOMETRY
Principle: Measures the current flow between two non-
polarizable electrodes between a known electrical
potential is established

Annie June Audan

 COULOMETRY - Electric neutrality; separation is strictly on the
Principle: Measures the amount of electricity basis of electric charge and uniformity of
(coulombs) at a fixed potential material size.
- 1 coulomb = 1 amper per second 5. POLYACRYLAMIDE ELECTROPHORESIS
- Uses the FARADAY’S LAW - Uses protein size as the major factor in the
o Number of coulombs consumed can separation process and the net charge of
be directly related to the proteins
concentration of the unknown - Another medium of choice
- Durable; stable
ELECTROPHORESIS
Principle: migration or movement of charged particles
in an electric field. New Approaches of Electrophoresis
- A charged particles or ion will migrate 1. High resolution Protein Electrophoresis
towards the anode or cathode depending on - Modified Electrophoresis technique that
the isoelectric pH of the soln under the permits the detection of 12 – 15 diff protein
influence of an externally electric field. fractions
- Modification: addition of Calcium chloride to
Factors Affecting Rate of Migration the buffer (pH 8.6), addition of colling system
Higher electric charge = and increased voltage for the separation
Net Electric Charge
faster migration causing no damage to the gel or protein
Size & Shape of Bigger molecule = slower 2. Isoelectric Focusing
Molecule migration - Concentrates on the isoelectric point of the
Higher ionic energy = particles and makes use of carrier ampholytes
Electric Fluid Strength for the detection of specific proteins
slower movement
More voltage = more - Used in the study for the detection of specific
movement isoenzymes
PROBLEM WITH 3. Two-dimensional Protein Electrophoresis
INCREASED TEMP - Combination of isoelectric focusing and
a. Denaturation of polyacrylamide gel electrophoresis that is
proteins (above capable of identifying most unique proteins
Temperature the cannot be measured by single techniques
40 C/ 105.8 F)
b. Evaporation of 4. Immunoisoelectrophoresis (IEP)
solvent - Separation of protein fractions based on
increases antigen-antibody reactions
strength of the
buffer PROTEIN DETECTION AFTER SEPARATION
Zone electrophoresis Techniques:
Nature of Supporting (charge molecules that 1. Colored dyes (for all proteins)
Media migrate to different 2. Enzyme reactions (for detection of specific
pores) enzymes)
3. Immunofixation (for specific proteins w/c are
Types of Supporting Media not enzymes)
1. PAPER ELECTROPHORESIS 4. Silver staining (useful for urine and CSF for the
- Earliest support media detection of extremely low levels of proteins)
- Paper is fragile and easily damaged staining Clearing Agents: it is used to dry up to the supporting
of protein medium thereby preventing the diffusion of the
separated fractions to the other fractions
2. STARCH GEL ELECTROPHORESIS Ex. Methanol, cyclohexanol, acetic acid, dioxane
- Good for large samples
- Fragile and unable to store results QUANTITATION OF SEPARATED PROTEIN
permanently FRACTIONS
1. Elution Technique followed by
3. CELLULOSE ACETATE ELECTROPHORESIS Spectrophotometry
- Strip with a clear plastic backing with a - Done by cutting the electrophoretic strip and
coating of cellulose acetate particles attached eluting each fraction into soln for further
to it chemical analysis; time consuming
- Treated with acetic anhydride 2. Densitometry (method of choice)
- Becomes brittle when dried - The measurement of density of light passing
4. AGAROSE ELECTROPHORESIS through the fraction (colorimetry); based on
- Mixture of polysaccharides from seaweed the amount of light absorbed w/c is roughly
- Medium of choice for chemistry proportional to the concentration of the dye
- Used in separation of serum, CSF, urine, and concentration of protein fraction.
lipoprotein
- Pores are large enough for all proteins to pass Specimens: Serum, Urine, CSF
through Clinical Use: analysis of proteins(serum) that can
provide quick and useful info regarding the
presence/absence of disease entities.

Annie June Audan

Components of Electrophoresis System:
Quality Assurance & Quality Control
1. Support media
2. Electrophoretic chamber
QUALITY ASSURANCE
3. Power supply
- Overall program that ensures that the final
results reported by the laboratory are correct
o Pre- analytic
o Analytic
o Post- analytic

AUTOMATION
- Mechanization of steps/procedures
Advantages
✓ Rapid results
✓ Increase number of tests performed
✓ Saves time and effort
✓ Eliminates the need for more staff
✓ Economical
✓ Reduces errors in calculation and transcription
✓ Better precision and accuracy
Basic Approaches in Automation
1. CONTINUOUS FLOW ANALYZER
- All samples are carried through the same
analysis pathway. All samples are
automatically pass from one step to another
w/o waiting to bring the samples to the stage
of completion.
- Sequential analysis (each samples pass the
same pathway)
- Uniformity in test performance
- Use in both routine and research purposes
• Technicon Autoanalyzer II – capable of
running 3 different tests at 60-80 samples/hr
2. DISCRETE ANALYZER
- Each sample reactions is handled in a
separate compartment and does not come
into contact with another sample.
- Separate analysis (each sample do not come
in contact with each other)
- Most popular and versatile
• Run multiple tests in one sample at a time or
multiple samples in one test at a time
3. CENTRIFUGAL ANALYZER
- As the rotor is accelerated, centrifugal force
moves the reagents and sample to a mixing
chamber and then trough a small channel into
the cuvette.
- Batch analysis (multiple sample)
- Centrifugal force moves the reagents and
samples to a mixing chamber into a cuvette,
passing a light beam and measuring the
absorbance
4. THIN FILM ANALYZER
- A 16 mm square chip w/c contains several
thin layers, accepts a metered drop of serum,
spreads it evenly onto a reagent layer, then
confines the colored product to a fixed area
for reflectance spectrophotometry.
- Ex. Kodak “EktaChem
QUALITY CONTROL
- System of ensuring accuracy and precision in
the lab
- Process of ensuring that analytical results are
correct
- QC samples are measured periodically in the
same manner as clinical samples
Kinds of QC
1. INTRALAB
- Internal QC
- Analyses of control samples together with
patient specimens
- Important in daily monitoring
2. INTERLAB
- External QC
- Involves proficiency testing programs
o National External Quality Assessment
Service (NEQAS) – mandatory to all
labs
- Maintain long-term accuracy of methods
Objectives of QC
✓ To check the stability of the machine
✓ To check the quality of the reagent
✓ To check for technical/clerical error
Characteristics of an Ideal QC Material
✓ Resembles human sample
✓ Inexpensive and stable for long periods
✓ No communicable diseases
✓ No matrix effects
✓ With known analyte concentrations
✓ Convenient packaging for easy dispensing
and storage
Types of Reagents
1. BLANK
- Reagents without analyte added
- Water; distilled water
2. STANDARD
- Most specific analytical soln
- Value will tell the concentration of the
unknown
- Only one analyte
• Each analyte has specific standard
3. CONTROL
- Value will determine accuracy and precision
- Resembles patient sample
- Many analytes
• Usually has 3 control reagents
o 1 = abnormally increased/high
o 2 = normal
o 3 = abnormally decreased/low

Annie June Audan

 SCREENING TEST DIAGNOSTIC TEST • False negative (FN) – Indicating a person does
Done on apparently Done on sick or ill not have the disease when, in fact, he or she
healthy individuals individuals does.
Not a basis for • True negative (TN) – Indicating a person does
Basis for treatment not have the disease when, in fact, he or she
treatment
Less accurate; less More accurate; more does not.
expensive expensive
STATISTICS
QUALITY CONTROL PARAMETERS Measure of central
MEAN
VALIDITY RELIABILITY tendency
Precision; MEDIAN Middle value
Accuracy Reproducibility; MODE Most frequent value
Repeatability Highest value – lowest
RANGE
How well the test value
How well the test STANDARD DEVIATION Measure of dispersion
measures what it is
performs in use over time
supposed to measure VARIANCE Measure of dispersion
How close is the result How close are the results COEFFICIENT OF
Index of precision
to its true value of a test on repetition VARIATION

TYPES OF ERRORS
SYSTEMATIC ERROR RANDOM ERROR
Predictable Error Unpredictable Error
An error that is constant Due to instrument,
when measurements are operator, and
made under the same environment conditions
condition
1. High accuracy & High precision = close to true Causes: deterioration of Causes: pipetting error,
value & close to each other reagents, improperly mislabeling of samples,
2. Low accuracy & High precision = far from true made standard temperature fluctuation,
value & close to each other solutions, contaminated improper mixing of
3. High accuracy & Low precision = close to true solutions, calibration sample and reagent
value & far from each other problems, failing
4. Low accuracy & Low precision = far from true instrumentation
value & far from each other Has a problem in Has a problem in
accuracy precision
SENSITIVITY SPECIFICITY
Ability of test to detect Ability of test to detect
the smallest amount of substances without
analyte interferences
Diagnostic Sensitivity - Diagnostic Specificity -
Ability of the test to Ability of the test to
identify correctly those identify correctly those
LEVEY – JENNINGS CHART
who have the disease who do not have the
- Most commonly used histogram in QC
(a) from all individuals disease (d) from all
Histogram is/are sheets of rectangular coordinate
with the disease (a+c) individuals free from the
graphing paper where data for sequential analysis are
disease (b+d)
plotted to locate the source of error.
[TP/ (TP+FN)] x 100 [TN/ (TN+FP)] x 100
Check sick people (TRUE Check people with no
Types of result
POSITIVE) disease (TRUE
1. In Control – QC falls in confidence limit
NEGATIVE)
2. Out of Control – control values fall outside the
The higher the The higher the specificity
confidence limit
sensitivity % = the lower % = the lower the FALSE
Types of Out of Control
the FALSE NEGATIVE POSITIVE
1. TREND
- Formed by the control that continue to either
increase or decrease for a period of 6 or more
consecutive days

• 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.

Annie June Audan

 2. SHIFT
- 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
R2sd – one control
exceeding the +2s and
another exceeding the
-2s

3. OUTLIER
- Values which are far from the main set of
values due to wild errors

WESGUARD MULTIRULE SYSTEM

- A “multi-rule” system developed by Dr. James 41sd – it is violated if
O. Westgard based on statistical concepts four consecutive IQC
- A combination of decision criteria or rules to values exceed the
assess if a system is in control same limit (mean
±1sd)

12sd – it is violated if the

IQC value exceeds the
mean by ±2sd
13sd – it is violated when
the IQC value exceed the
mean ±3sd

10x – this rule is

violated when 10
consecutive IQC
values are on the
22sd – it detects same side of the
systematic errors and mean or target value
is violated when two
consecutives IQC
values exceed the
mean on the same
side of the mean by
±2sd

Annie June Audan

 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
1. 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
o Galactose
2. Disaccharides
- two monosaccharides are joined by a glycosidic
linkage
o Sucrose: Fructose + Glucose
o Lactose: Galactose + Glucose
o Maltose: Glucose + Glucose
3. 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
o After 30 mins – glucose levels rise
GLUCOSE METABOLISM
o After 1 hour – glucose levels peak
• As food enters the mouth and oral cavity, food
o After 2 hours – glucose levels will decrease
begins to be broken down by ptyalin, an enzyme
and go back to normal
produced by the parotid gland that helps in the
initial metabolism of food
GLUCOSE METABOLIC PATHWAY
• When food reaches the stomach, the acidity
1. Embden-Meyerhoff Pathway – provides energy for
inactivates ptyalin and acid hydrolysis occurs.
the body
There is no carbohydrate digestion in the stomach,
o Aerobic – glucose to pyruvate
but protein digestion happens through the enzyme
▪ Cells have O2
pepsin.
o Anaerobic – glucose to lactate
• When food reaches the intestines, amylopsin, an
▪ O2 in the body is used up
enzyme produced by the pancreas, further
(lack of O2 in the cells)
degrades the food and convert polysaccharides
usually after exercise
into monosaccharides.
▪ Lactate will seep in the
• Glucose then enters the bloodstream and
muscle resulting to a sore
increases glucose uptake by the cells.
feeling
2. Hexose – Monophosphate Shunt – production of
reduced NADPH and ribose-5-phosphate
3. Glycogenesis – glucose to glycogen

Annie June Audan

 Processes in Regulating Glucose Metabolism
• Glycolysis – glucose to pyruvate(aerobic) or
lactate(anerobic) to produce energy
• Glycogenolysis - breakdown of glycogen to
glucose for energy
• Gluconeogenesis – formation of glucose-6-
phosphate from non-carbohydrate sources
o Carbs are depleting in the
body/blood
• Glycogenesis – glucose to glycogen for storage
• Lipogenesis – conversion of carbohydrates to
fatty acids
• Lipolysis – decomposition of fat
Glucose is stored in the muscles and liver for
short term storage. In fats for long term.
Pancreas – organ releasing hormones; vital organ
Hormones – substances that have target cells to
perform specific function; released by specific organ
Endocrine Gland: release of hormones (insulin,
glucagon, somatostatin)
o Islets of Langerhans
Exocrine Gland: produces enzyme (amylase to
breakdown complex carbohydrates)
o Acinar and duct tissue
HORMONES REGULATING GLUCOSE METABOLISM
• Insulin (Glucose IN cell)
- produced by the β cells of the islets of
Langerhans in the pancreas
- promotes the entry of glucose inside the cell
- only hormone that decreases glucose
(hypoglycemic hormone)
- promotes glycolysis, glycogenesis, lipogenesis
& protein synthesis
* without insulin, the glucose will just stay in the
bloodstream; thus increasing blood sugar level
* high glucose level in the blood (stimuli) will
trigger the production of insulin
o Proinsulin: immediate precursor of insulin
▪ Pre insulin → prepro insulin
→ pro insulin → insulin
▪ big molecule that will cleave
into 3
o 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
o Cleaved portion of cleaved
proinsulin
o Detectable in DBM2 because the
person can still produce insulin
unlike in DBM1 where there is no
insulin production due to auto
destruction of beta cells
• Glucagon
- produced by the α cells of the islets of
Langerhans in the pancreas
- primary hormone for increasing glucose levels
- major hyperglycemic hormone
*stimuli: decrease/low glucose level
- promotes glycogenolysis and gluconeogenesis
& ketogenesis
- influences hepatic glycogenolysis
• Somatostatin
- produced by the δ (delta) cells of the islets of
Langerhans in the pancreas
- inhibition of pancreatic hormone release of
insulin and glucagon (to prevent excess
production after body reaches equilibrium)
- serves as a regulatory hormone
- inhibition of gastric acid secretion
OTHER HORMONES THAT CAN INCREASE GLUCOSE
LEVEL
• 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 (T3 & T4)
- produced by the thyroid gland
- promotes glycogenolysis and intestinal
absorption of glucose
• Growth hormones
- produced by the anterior pituitary gland
- promotes glycogenolysis and lipolysis
• Adrenocorticotropic hormone (ACTH)
- produced by the anterior pituitary gland
- promotes glycogenolysis and gluconeogenesis
Hypoglycemia
- Low blood glucose levels
- Imbalance glucose utilization and production
(not a usually a disease state)
- May be due to insulinoma or diabetic shock
• Fasting Hypoglycemia – very depleted glucose
in the body (due to activity or disease state)
o not eating for a long time but doing
tons of activities
o alcohol induced fasting hypoglycemia
(when you drunk to much alcohol and
you slept for too long)
o Hepatic diseases; endocrine diseases
(insulinoma- tumor found in the
pancreas that leads to hypersecretion
of insulin);
• Reactive Hypoglycemia – following a stimulus
Annie June Audan
 o Post prandial hypoglycemia after
gastric surgery
o Drug induced (e.g for galactosemia
meds)
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 (light headed, weak,
trembling hands, numbness, perspiration,
fatigue, hungry & emotional: HUNGRY)
2. Low blood glucose
3. Relief of symptoms when glucose is raised to
normal
Hyperglycemia
- high blood glucose levels (FBS >126mg/dL)
- Causes: stress, severe infection, dehydration
or pregnancy, pancreatectomy(removal of
pancreas), insulin deficiency or abnormal
insulin receptor
o Dehydration; the glucose in blood is
too concentrated due to lack of
water/fluid (Pseudodilutional)
o Pancreatectomy – no more hormone
that can decrease the glucose, unlike
the increase of glucose since there
are a lot of other hormones that can
influence it
- S&S: fatigue, blurry vision, excessive thirst
(polydipsia), increased hunger (polyphagia),
nausea & vomiting, polyuria, fruity breath
- Ketones – another source of energy; come
from lipids; it has 3 types:
o Acetoacetate,
o B-Hydroxybutyrate,
o Acetone – escape as a gas; resulting
to a fruity breath (sugar)
Laboratory findings
o increase glucose in plasma and urine
o increase urine specific gravity (increased
amount of solute in urine)
o increase ketones in serum and urine
o increase osmolality
o decrease blood and urine pH
o electrolyte imbalance
- Values greater than 500 mg/dL may result to
multiple organ failure
- microvascular complication of hyperglycemia:
o nephropathy – kidneys; leading cause
of end-stage renal disease
o neuropathy – nerves; nontraumatic
lower extremity amputation (beware
of the feet)
o retinopathy – eye; leading to
blindness
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
- Due to glucose being an osmotic
molecule (increase fluid intake,
increase voiding)
- Osmotic diuretic
o Polydipsia- excessive thirst
- Due to increase voiding/urinating
o Polyphagia- increased food intake
• Glucosuria is the presence of glucose in the urine
* glucose should not be in the urine because it is a BIG
molecule
* renal glucose threshold = 160-180mg/dL; kidney’s
limit for glucose reabsorption; excess will be excreted
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
- autoimmunity
- caused by the cellular-mediated autoimmune
destruction of the beta cells of the islets of
Langerhans in the pancreas (no more insulin
production, the glucose will stay in the
bloodstream)
*body will use protein and lipids in place of carbs; will
produce ketones
Ketones – another source of energy; come from lipids
- juvenile onset: 5-10% affected
- Signs And Symptoms: Rapid weight loss (the
body cannot utilize carbs; using lipids and
proteins for energy steal what’s meant for
other parts of the body), hyperventilation,
mental confusion, possible loss of
consciousness
- Prone to ketoacidosis (DIABETIC
KETOACIDOSIS)
- C-peptide: Undetectable to very low
- Treatment or management include Exogenous
Insulin Therapy
Annie June Audan
 TYPE II DIABETES MELLITUS
- also known as Non-insulin dependent DM,
Stable diabetes, Adult onset DM
- no autoimmunity
- caused by insulin receptor defect leading to
insulin resistance
- there is no problem with insulin production;
insulin receptor is the problem since it can’t
detect insulin;
- it will not use other sources of energy because
not all cells are defective
Insulin receptors – found in the cells to attract and bind
with insulin to allow the entrance of glucose
- adult onset: 90-95% affected (developed
overtime; diet & lifestyle)
- Signs And Symptoms: Weight gain (influenced
by lifestyle), milder symptoms than Type I DM
- Not prone to ketoacidosis
o Not all of the cells are resistant to
insulin
o There are still normally functioning
insulin receptors so there are cells
that allows glucose intake
- C-peptide: detectable/high
- Management: Healthy lifestyle
o Excessive carbs & sweets
consumption will result to excess
glucose in the blood because the
blood cells can’t intake every glucose
as there are some that are resistant
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
hyperbilirubinemia, overweight, congenital
malfunctions/malformations
- Screening should be performed between 24th
and 28th weeks of gestation (1-hr Glucose
Challenge Test using 50g glucose load or
OGTT)
- Macrosomia – fetus larger than average; more
than 8 pounds
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:
o 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
- Specimens:
o whole blood – only in HbA1c
o serum –
o plasma -
o CSF – diagnosis for meningitis
o Urine
o Synovial fluid
Fasting Blood Sugar (FBS)
- screening test for DM
- detect elevation of blood glucose
- 8-10 hours (NPO) fasting is required; not more
than 16hrs (over fasting)
Categories of fasting plasma glucose
- Normal = <110 mg/dL
- Impaired fasting glucose = >110- <126 mg/dL
- Provisional Diabetes dx = >126 mg/dL
1. Random Blood Sugar (RBS)
- sugar determination of randomly collected
blood
- requested during insulin shock or
hyperglycemic ketonic coma
2. 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
3. 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 (can walk); 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. Repeat if the mother puke.
Annie June Audan
 ✓ RECORD the time the mother finished drinking
everything.
Categories of OGTT:
- Normal: 1 hr plasma glucose <140 mg/dL
- Impaired glucose tolerance: >140-<200 mg/dL
- Provisional diabetes diagnosis: >200 mg/dL
4. Intravenous Tolerance Test (IVTT)
- for those who cannot take large carbohydrate
intake, altered gastric physiology, undergone
operation in intestine, chronic malabsorption
syndrome
5. Insulin Tolerance Test (ITT)
- evaluates sensitivity to insulin
- glucose is administered orally 30min after
insulin
6. Insulin glucose tolerance test
- Glucose is administered orally 30 mins after
insulin administration
7. Other Tolerance Test
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
8. Glycosylated Haemoglobin Test
- Gold standard test for glucose (can’t be
cheated)
- HbA1c = Hb – hemoglobin; A1c – fragment of
beta cell
- 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 (hemolytic anemia)
- Specimen: Whole blood EDTA
- Uses column chromatography or
electrophoresis
- Normal value: < 6.5%
- (5.7-6.4% = prediabetes)
- For every 1% increase in GHb, there is 25
mg/dL change in plasma glucose
9. Fructosamine
- Glycated albumin; plasma protein ketoamine
- Fructosamine is formed by attachment of
glucose to albumin
- Short term monitoring of glucose
- Detects glucose levels in the average of 2-3
weeks since the lifespan of albumin in the
circulation is 20 days
- Not suitable for patients with
hypoalbuminemia
- Specimen: Serum
- Normal value: 205-285 μmol/L or 2-2.9%
METHODS FOR GLUCOSE TESTING
Copper Reduction Methods (oldest method)
- Principle: glucose in a hot alkaline solution
readily reduces cupric ions to cuprous ions
1. Folin Wu Method
Cuprous + phosphomolybdate = phosphomolybdenum
blue
- Disadv: non-glucose reducing sugars are not
precipitated during process
2. Nelson Somogyi Method
Cuprous + arsenomolybdate = arsenomolybdenum
blue
- Adv: non-glucose reducing sugars are
adsorbed by barium sulfate
3. Neocuproine Method
Cuprous + Neocuproine reagent = yellow-orange
complex
- Reagent: Neocuproine rgt (2,9-dimethyl-1,10-
phenanthroline HCl
4. Shaffer-Hartman Somogyi Method
Cuprous + Iodine + Acidic solution = colorless complex
- excess iodine is titrated with thiosulfate
5. Benedict’s Method (modified Folin Wu
method)
Copper sulfate (blue) + glucose + heat = brick red
precipitate
Alkaline Ferric Reduction Method
1. Hagedorn-Jensen Method
Ferricyanide (yellow) + glucose = ferrocyanide
(colorless)
- inverse colorimetry; measured at 420 nm
- widely used in Technicon Auto Analyzer
system
Condensation Method
1. Condensation with Phenols
aldehyde group + phenol = colored complex
- Common reagents: anthrone, thymol,
phloroglucinol, resorcinol, alpha naphthol, m-
aminophenol
Annie June Audan
 2. Condensation with Aromatic Amine Home Monitoring Glucose Method:
- Common reagents: o-toluidine, aniline, 2- 1. Dextrostics (cellular strip)
aminophenyl - using only a drop of capillary blood, the
reaction on the reagent pad is read by
Dubowski Method (o-Toluidine Method) reflectance colorimeter.
glucose + 0-toluidine + acetic acid + heat = green - glucose value of > 140 mg/dL should be
complex (630 nm) rescreened with FBS, HbA1c, or OGTT using
- most specific non-enzymatic method for venous samples.
glucose - should not be used to diagnose diabetes or
other hyperglycemic conditions.
Enzymatic Methods -
1. Glucose oxidase method (GO) (oldest method) Inborn Errors of Carbohydrate Metabolism
- Principle: glucose exists as α (35%) or β (65%) • GLYCOGENOSES = defect in glycogen
mutarotase converts alpha to beta glucose metabolism
which results to glucose oxidase specific to • consequence of inherited deficiencies of
beta glucose. enzymes that control the synthesis or
- False results: increased ascorbate, bilirubin, breakdown of glycogen
uric acid, glutathione, creatinine, l-cysteine, l- • Can either cause abnormal quality or quantity
dopa, dopamine, methyldopa, and citric acid of glycogen
because it ingibit peroxidase • Liver (hypoglycemia and hepatomegaly) and
muscles (muscle cramps, exercise intolerance,
2. Saifer-Gernstenfield Method (Trinder fatigue, weakness) are commonly affected
Reaction) (Colorimetric)
glucose + O2 → gluconic acid + H2O2 Genetic
TYPE DEFECT
H2O2 + chromogenic subs → H2O + oxidized s
chromogen Type I Von Autosom
Glucose – 6 – al
- coupled enzymatic method ( GO and (liver) Gierke
phosphate recessive
Peroxidase) disease
- chromogen: O-toluidine (yellow), O-dianisidine Type II N/A
(orange, o-indophenol (blue) Pompe Lysosomal acid
(muscl
disease α – glucosidase
e)
3. Polarographic Type III Cori or Glycogen Autosom
- The oxygen consumed in the oxidation (liver) Forbe’s debranching al
process of glucose tp gluconic acid is disease enzyme recessive
measured using polarographic oxygen Type Autosom
electrode. Anderson Glycogen al
IV
- oxygen consumption is directly proportional to ’s disease branching enzyme recessive
(liver)
glucose concentration Type V N/A
glucose + O2 → gluconic acid + H2O2 McArdle Muscle
(muscl
H2O2 + C2H5OH → CH3CHO + 2H2O disease phosphorylase
e)
H2O2 + 2H + 2I → I2 + 2H2O Type Glycogen Autosom
- add molybdate, catalase, iodine, and ethanol Hers al
VI phosphorylase
disease recessive
(liver) (liver)
4. Hexokinase method (highly specific and Type N/A
reference method) VII Tarui Phosphofructokin
- False decrease results: hemolyzed and icteric (muscl disease ase
spx e)
Glucose + ATP → Glucose-6-phosphate + ADP Type X-
G-6-P + NADP →6-phosphogluconolactone + Phosphorylase
IX Linked
NADPH kinase (liver)
(liver)
- coupled enzymatic method (G6PD and Autosom
Type Fanconi –
Hexokinase) al
XI Bickel GLUT 2
(liver) disease recessive
Type 0 Glycogen
(liver) Synthase

Annie June Audan

 LIPIDS AND LIPOPROTEINS
▪ Lipids are basically fats.
▪ Lipid’s solubility is always opposite to water
solubility
▪ Lipid soluble substances are insoluble to water
▪ Lipid insoluble substances is soluble in water
▪ Fatty acid is the main component of lipids
NOTE: If a substance is water insoluble, it will have
difficulty entering the bloodstream, thus the need for
carrier protein
BIOLOGICAL FUNCTION OF LIPIDS
1. Fuel or energy
- Back up only since carbs are the main
2. Insulation
- Fats are bad conductor of heat
3. Protect or cushion internal organs
- Lipids wrapped internal organ to protect
from trauma; padding
4. Building blocks of other substances in the body
- Steroid hormones are basically made up of
lipids (cholesterol)
5. Constituent of cell walls
- Cells are made up of phospholipid bilayer
(hydrophilic and hydrophobic heads)
LIPID CHEMISTRY
Triglycerides (triacylglycerol) (TAG)
- major class of dietary lipids
- glycerol + 3 fatty acid chains
- comprises 95% of lipids in food and human
body
- present in dietary fat
- synthesize by liver and adipose tissues for
back up energy
- FATS: lipids that are solid at RT (saturated
fatty acids from animals)
- OILS: lipids that are liquid at RT (unsaturated
fatty acids from plants)
Phospholipids
- constituent of cell membranes
- bilayer sheet
- head: hydrophilic
- tail: hydrophobic
FORMS:
- 70% Lecithin/Phosphatidyl choline
- 20% Sphingomyelin
- 10% Cephalin
Cholesterol
- precursor of steroid hormones (testosterone;
other adrenal hormones)
- not used for energy
- can’t burn or sweat cholesterol
- the only hydrophilic part of cholesterol is the
hydroxyl group in the A-ring
- amphipathic lipid
- 90% endogenously (not affected by diet)
produced in liver
- Esterified form: cholesteryl ester
▪ Neutral lipids
▪ Found at the center of lipid drops and
lipoproteins along with TAG
- Phytosterols (sterols) – cholesterol
synthesized by plants
- A small amount of cholesterol, after first being
converted to 7-dehydrocholesterol, can also
be transformed to vitamin D3 in the skin by
irradiation from sunlight
- A small amount of cholesterol can also be
converted by some tissue, such as the adrenal
gland, testis, and ovary, to steroid hormones,
such as:
▪ glucocorticoids,
▪ mineralocorticoids
▪ estrogens
Fatty Acids
- building blocks of lipids
- only a relatively small amount of fatty acids
exists in the free or unesterified form
- majority of plasma fatty acids are instead
found as a constituent triglycerides or
phospholipids
- elaidic acid: major dietary trans fatty acid
- most fatty acids are synthesized in the body
from cholesterol precursors except:
▪ linoleic
▪ linoleic acid
Annie June Audan
 Chylomicron
- Gamma (γ) lipoprotein
- Chylo- milky or juicy fluid; micron- small
particles
- Largest and least dense
- Produced in the intestine
- Contains: the most TAG
o 85-92% TAG
o 6-12% phospholipids
o 1-3% cholesterol
o 1-2% proteins
- Exogenous Pathway (outside of the body)
- function: transport TAG absorbed from the
intestine to adipose, cardiac & skeletal muscle
- TAG components are hydrolyzed/metabolized
by lipoprotein lipase thereby releasing FFA to
NOTES: Ketones are formed when: the tissues
1. Prolonged starvation - Chylomicron remnants are taken up by the
2. Problem in carbs metabolism liver, thereby transferring dietary fat to the
Ketones are derived from fatty acids. It is reversible liver
once carbs are utilized again. - Responsible for post-prandial turbidity or
It has 3 types of bodies milky appearance of sample
1. Acetone - Pro-atherogenic (can contribute to the fatty
2. Acetoacetic acid (ketoacidosis) deposits in the arteries leading to
3. Beta-hydroxybutyric acid (beta- atherosclerosis)
hydroxybutyrate)

LIPOPROTEINS
▪ Lipids + proteins
▪ bound to the proteins which allow fats to move
through the water in and out of cells
▪ Function: emulsify lipid molecules

Composition of Lipoproteins
1. Chylomicrons
2. Very Low Density Lipoprotein
3. Intermediate Density Lipoprotein
4. Low Density Lipoprotein
5. High Density Lipoprotein

▪ Most dense to the least (based on density):

HDL > LDL > VLDL > Chylomicron

▪ Heaviest to lightest (based on Molecular Weight):

Chylomicron > VLDL > LDL > HDL

NOTE: The smaller the molecule is, the denser it will be.
The larger the lipoprotein is, the lighter it is in density.

Annie June Audan

LPL- lipoprotein lipase an enzyme that breaks down
the TAG inside the chylomicron to release free fatty
acids (FFA) to be absorbed by the muscles and
adipose tissue
Two sources of TAG
1. Endogenous – from body
2. Exogenous – from diet
Very Low Density Lipoprotein (VLDL)
▪ Pre-beta lipoprotein
▪ Lipoprotein made in the liver
▪ Second highest TAG content
▪ Endogenous pathway of TAG
▪ function: Endogenous transport of TAG;
body's internal transport mechanism for lipids
▪ from the liver to muscle and adipose
▪ Responsible for fasting hyperlipidemic
turbidity of sample (liver endogenously
produces TAG despite px is fasting)
▪ Pro-atherogenic
TAG is synthesized in the liver
Intermediate Density Lipoprotein (IDL)
- VLDL remnant
- similar to LDL wherein it transports TAG, fats
and cholesterol and can also promote the
growth of atheroma
▪ go back to the liver for further
synthesis
- function: enables fats and cholesterol to move
within water-based solution of the
bloodstream
- Pro-atherogenic
TAG – chylomicrons & VLDL
CHOLESTEROL – LDL & HDL
Low Density Lipoprotein (LDL)
- Beta lipoprotein
- products of VLDL and IDL metabolism
- most cholesterol-rich
- function: principal cholesterol and fat
transport in blood; carries chole form the liver
to tissue and cells
- known as the “bad cholesterol” because it is
involved in the progression of cardiovascular
disease (CVD) like atherosclerosis or stroke
- responsible in depositing cholesterol in arterial
lining and peripheral tissues
- macrophages that take up too much lipid
become filled with intracellular lipid drops and
turn into foam cells
- pro-atherogenic
- metabolism: 40-60% cleared by hepatic LDL
receptors; the rest are taken up by non-
hepatic (scavenger) receptors such as
macrophages
- INTRACELLULAR CHOLESTEROL
TRANSPORT
▪ From the liver to cells
High Density Lipoprotein (HDL)
- Alpha lipoprotein
- smallest and densest
- most abundant in apolipoproteins
- phospholipid is the main lipidic content
- discoidal
- function: reverse cholesterol transport;
transport of excess cholesterol from tissues
and cells back to the liver
- hepatocytes have receptors from APO A1 →
HDL that absorbed excess cholesterol from
the tissues are then taken up by the
hepatocytes
- known as the “good cholesterol” because it
protects against heart disease
- metabolism: synthesized in the liver and
intestine
- discoid shape to absorb cholesterol then
become spherical in shape
- protects against heart disease
- Reverse Cholesterol Transport
▪ From the cells to the liver
Annie June Audan
 LIPOPROTEIN VS APOLIPOPROTEIN

Lipoprotein Apolipoprotein
Any of a group of The proteins that bind
soluble proteins that lipids (oil-soluble
combine with and substances such as fat
transport fat or other and cholesterol) to form
lipids in the blood lipoproteins
plasma
Synthesized in the liver Synthesized both in the
Apolipoproteins intestine and liver
▪ Apolipoproteins are proteins that bind lipids to form Compose of a Compose of proteins
lipoprotein phospholipid and bound to phospholipids
▪ Found in the surface of lipoproteins cholesterol outer shell
▪ They serve as enzyme cofactors and receptor and a hydrophobic core
ligands of cholesterol esters and
▪ They regulate the metabolism of LP and their uptake TAG
in tissues Classes: HDL, LDL, IDL, Classes: apolipoprotein,
ApoLP LP CARRIED/FUNCTION VLDL, and ULDL A, B, C, D, E, H, L, and
Main protein in HDL apolipoprotein (a)
Apo A-1
Activator of LCAT for esterification Serves as a carrier Serves as an structural
Inhibits HGTL at high concentration molecules for the component in
Apo A-II
Structural receptor binding of HDL transport of hydrophobic lipoproteins, ligands for
Structural receptor binding of VLDL, lipids through the blood the surface receptors,
Apo B-100 LDL and cofactors for
Critical in uptake of LDL by cells enzymes
Structural receptor binding of LDL increases the risk of Apolipoprotein B-100
Apo B-48
Chylomicrons coronary heart disease increases the risk of
Lipemia clearing factor CHD
Apo C-II Activates LPL, targets TAG and HDL lowers the risk of Apolipoprotein A-1
removes CM after meal CHD lowers the risk of CHD
Inhibits LPL, inhibits clearance of CM
Apo C-III
after meal
VLDL, LDL, HDL LIPID STORAGE DISEASES
Apo E
Increased in Alzheimer’s Dx
Accumulation of sphingomyelin
NIEMANN-PICK
in the bone marrow, spleen
DISEASE
and lymph nodes
Complete absence of HDL
(HDL: 1-2 mg/dL)
TANGIER’S
Clin cx: orange or yellow
DISEASE
discoloration of tonsils and
pharynx
Deficiency of hexosaminidase
TAY-SACH’S A
DISEASE Accumulation of sphingolipids
in the brain
ANDERSON’S Chylomicron-retention disease
DISEASE
Plant sterols absorbed and
accumulated in the blood and
SITOSTEROLEMIA
tissues
Consuming plant based food

Annie June Audan

 Lipid Disorders

TAG CHOL CM LDL VLDL

LPL deficiency (Low cardiac

1: Hyperchylomicronemia risk, eruptive xanthoma, ↑ N ↑ N N
recurrent pancreatitis)
Defective or deficient LDL
receptors (High cardiac risk,
2a: Familial xanthelasma, tendon
N ↑ N ↑ N
hypercholesterolemia xanthoma, corneal arcus,
hypothyroidism, nephritic
syndrome)
2b: Familial combined Most common
↑ ↑ N ↑ ↑
hyperlipidemia (High cardiac risk)
3: Familial Accumulated VLDL (Eruptive
↑ ↑ N N ↑
dysbetalipoproteinemia and palmar xanthomas)
4: Familial Low cardiac risk
↑ N N ↑
hypertriglyceridemia
Low cardiac risk, eruptive
5: ↑ ↑ N ↑
xanthoma
Defective apo β synthesis
Abetalipoproteinemia/
(Cerebral ataxia,
Bassen-Kornzweig ↓ ↓ Not found in Plasma
acanthocytosis, fat
Syndrome
malabsorption)

INCREASED
Type TAG Cholesterol Standing plasma test
PARTICLE
Type 1 – Familial Chylomicronemia or LPL Positive,
CM High Normal
deficiency Clear plasma
Negative,
Type 2A – Familial Hypercholesterolemia LDL Normal High
Clear Plasma
Type 2B – Combined VLDL Negative,
High High
hyperlipoproteinemia LDL Cloudy Plasma
Occasional cloudy
Type 3 - Dysbetalipoproteinemia IDL High High
plasma
Negative,
Type 4 – Primary Hypertriglyceridemia VLDL High Normal
Cloudy Plasma
VLDL Positive,
Type 5 – Mixed hyperlipidemia High High
CM Cloudy Plasma

RISK FOR CORONARY HEART DISEASE

Age Bracket CHOLESTEROL LEVEL (mg/dL)
Moderate Risk High Risk
2-19 yrs old >170 >185
20-29 yrs old >200 >220
30-39 yrs old >220 >240
>40 yrs old >240 >260

SPECIMEN CONSIDERATIONS ▪ LDL determination (computed)

- Requires at least 10-16 hours of fasting ▪ VLDL determination (computed) (indirectly
▪ At least 10 hrs if fbs and lipid profile are measured)
together Note: not affected by fasting:
- Not affected by fasting: CHOLESTEROL - Cholesterol determination since 90% are
determination endogenously produced
- Hemolyzed and icteric samples are not accepted
- Specimen: Serum or plasma EDTA Friedewald Equation
▪ Best to use if serum - Computation to compute lipoproteins
- Commonly used
TC = HDL + LDL + VLDL
LIPID PROFILE TEST VLDL= TAG/5 or TAG/2.175
▪ Triglyceride determination LDL = TC – HDL – TAG/5 (mg/dL)
▪ Total Cholesterol determination (TC) LDL = TC – HDL – TAG/2.175 (mmol/L)
▪ HDL determination

Annie June Audan

 De Long Equation
TC = HDL + LDL + VLDL
LDL = TC – HDL – TAG/ 6.5 (mg/dL)
LDL = TC – HDL – TAG/2.825 (mmol/L)

CHOLESTEROL MEASUREMENT
▪ Abell-Kendall Method (OLD reference method)
- 3-step method: Saponification -> Extraction ->
Colorimetry
- Libermann-Burchard rgt: gHAc, acetic
anhydride, concentrated H2SO4
- End Product: cholestadienyl monosulfuric acid
- End Color: GREEN

▪ Abell, Levey, Brody Method (Current Reference

Method)
- Reagents: Libermann-Burchard rgt + KOH and
alcoholic hexane
- End Color: GREEN

▪ Sperry Method
- 4-step method: Saponification -> Extraction ->
Colorimetry -> Purification with digitonide

▪ Salkowski Method
- End Product: cholestadienyl disulfuric acid
- End Color: RED
▪ Enzymatic Method (most commonly used)
- interferences: ascorbate, hemoglobin, bilirubin
▪ Isotope Dilution/Mass Spectrometry (IDMS)
- Definitive method
▪ Electrophoresis
- Preferred gel: agarose gel
- From origin to the most anodal: Chylo, LDL,
VLDL, HDL
- Lipid-staining dyes: Oil Red, Fast Red 78,
Sudan Black, Scharlach Red

TRIGLYCERIDE MEASUREMENT
▪ Van Handel & Zilversmit Method
- Colorimetric Method
- Reagent: chromotropic acid
- End Color: BLUE
▪ Modified Van Handel & Zilversmit Method
- Colorimetric Method and Reference Method
- Reagent: sulphuric acid, salicylic acid
- End Color: PINK

▪ Hantzsch Method
- Fluorometric method
- Reagent: acetylacetone (diacetyl acetone)
- End Color: YELLOW

LIPOPROTEIN ANALYSIS
▪ Ultracentrifugation
- Reference method
- Measured in Svedberg units
- Based on density
- Reagent: potassium bromide w/ 1.063 density
- Fluorometric method
- Floating layer: Chylo, VLDL (yellow)
- Regeant (clear)
- Sinking layer: LDL, HDL (yellow)
DENSITY LIPOPROTEINS
<0.96 Chylomicrons
<1.006 VLDL
1.006 – LDL
1.063
1.006 – 1.21 HDL

Annie June Audan

 PROTEIN DIGESTION
PROTEINS

▪ The word PROTEINS come from the Greek word

“proteis” which means “first rank of importance”
▪ Most proteins are synthesized in the liver except:
o Immunoglobulins (plasma cells),
o adult hgb (bone marrow)
o factor VIII (included in coagulation cascade;
produced by endothelial cells)
▪ High molecular weight organic compounds compose
of amino acids combined together by peptide bonds
▪ MACROMOLECULE Pepsin – enzyme that breaks down protein into smaller
▪ Proteins are amphoteric which means that they are polypeptide
able to react both as a base and as an acid HCL – helps in protein denaturation to go to small
▪ Play vital role since it is involved almost in every intestine
body processes Enteropeptidase – convert trypsinogen to trypsin
▪ Carbon, Hydrogen, Oxygen, Nitrogen (CHON) Chymotrypsin – cleaves peptide bonds to convert into
specific amino acids for further protein synthesis in the
SIMPLEST FORMS liver
▪ CARBOHYDRATES – monosaccharides
▪ LIPIDS – fatty acids TOTAL PROTEIN – ALBUMIN = GLOBULIN (TPAG)
▪ PROTEINS – amino acids - Most common test for proteins
- Subtract TP – albumin = globulin

Protein Classification
▪ Primary Structure
- linear sequence of amino acids
- it determines the identity of protein, molecular
structure, function and binding capacity
- represents the number and types of amino
acids in the specific amino acid sequence

Biological function of proteins:

- Structural support for the tissue (collagen and
keratin) ▪ Secondary Structure
- Contraction and relaxation of muscles - refers to specific regular three-dimensional
- Coagulation and immunologic function formations into which portions of the
- Transport of metabolic substances polypeptide chain fold
(lipoproteins for lipids) - commonly formed structures stabilized by
- pH buffer hydrogen bonds between the amino acids
- Precursor of hormones (most of the hormones within the protein
are made up of proteins)
- Maintenance of osmotic pressure (influenced
by albumin)
- Biochemical catalysts (all enzymes are
proteins)

▪ Tertiary Structure
- actual 3-dimentional structure or folding
pattern
- responsible for physical and chemical
properties of proteins
- refers to the overall shape, or conformation
(fold), of the protein molecule

Annie June Audan


▪ Quaternary Structure
- association of 2 or more polypeptide chains
- the shape or structure that results from the
interaction of more than one protein molecule,
or protein subunits, held together by
noncovalent forces such as hydrogen bonds
and electrostatic interactions.
POLYMER OF AMINO AIDS
Structural Classification of Proteins
▪ Simple Proteins
- contain peptide chains which on hydrolysis
will yield only amino acids
- globular or fibrous in shape
▪ Conjugated Proteins
- contain protein and non-protein group
- Ex. Metalloproteins, lipoproteins,
glycoproteins, nucleoproteins
Types of Plasma Proteins
- Proteins found in plasma
Prealbumin (transthyretin)
- transport protein bound to thyroxine (T4) and
Retinol (Vit A)
- best quantified by immunologic
measurements since it is below the level of
detection by electrophoresis
Clinical Significance:
- Marker for nutritional status
- Confirms if specimen is CSF
o Since prealbumin is abundant in CSF
o Albumin is still most abundant in CSF
Albumin
- most abundant protein generally used for
transport
- abundant since fetal life
- accounts for half (50%) of all plasma proteins
- maintains fluid balance in tissues since it
influences 80% of the oncotic pressure
- major determinant of extracellular fluid
between intra and intervascular
units/compartments
- carry most of biological substances/molecules
(taxi of the body)
Clinical Significance:
- negative acute phase reactant (APR)
o the levels of substance will decrease
in cases of inflammation
o APR or positive APR are substances
that are related to inflammation
o PAPR substances will increase in
cases of inflammation
- decreased levels: malnutrition, malabsorption,
liver disease, renal disease, skin loss, dilution
- hyperalbuminemia – artifacts only; does not
mean that there is a disease; NOT an actual
disease state
o over infusion of albumin
o result of dehydration
o something is wrong in blood
collection
- hypoalbuminemia – an actual disease; it is not
good for albumin to decrease since it has been
the most abundant
NOTES:
- protein production is constant
- no such things that albumin will double in
synthesis
Globulins
- alpha 1, alpha 2, beta and gamma fractions
- differentiated based on migration
- dependent on electrophoretic mobility and
migration
α-1 antitrypsin (Anti-proteinase)
- major component of α-1 globulins
- inactivate proteases
o trypsin
o neutrophil elastase (released by WBC
to fight infection but it can also attack
normal tissues esp. in the lungs)
Annie June Audan
Clinical Significance: Clinical Significance:
- acute phase reactant - Acute phase reactant
- deficiency: pulmonary emphysema (chronic - increased levels: Menke’s Kinky Hair
obstructive lung disease), hepatic cirrhosis Syndrome
(genetic mutations) - decreased levels: Wilson’s disease (WD),
malnutrition, malabsorption
α-1 antichymotrypsin o WD- increased copper level in the
- serine proteinase inhibitor body and ceruloplasmins are all used
o chymotrypsin up by copper but still no equilibrium
- liver related ▪ Hallmark: Kayser Fleischer
Clinical Significance: Rings (dark rings that appear
- acute phase reactant to encircle the iris of the eye)
- associated with: Alzheimer disease and
Parkinson’s disease Haptoglobin
- Hgb binding protein
α-1 fetoprotein (α fetoprotein) - binds w hgb released by lysis of senescent
- protects fetus from immunologic attack by the RBC
mother - removed from the circulation by
- levels decrease gradually after birth reticuloendothelial system
- almost undetectable during adulthood - does not bind with myoglobin
Clinical Significance: Clinical Significance:
- increased levels: Spina Bifida, Neural Tube - acute phase reactant
Defects(spinal cord hindi mag close ng - evaluates degree of intravascular hemolysis
maayos), Anencephaly(undeveloped skull) o hemolytic transfusion reaction (HTR)
- decreased levels: Down Syndrome and o hemolytic disease of the newborn
Trisomy 18 (HDN))
- tumor marker in adults if detectable: - increased levels: Myoglobinuria
hepatocellular carcinoma - decreased levels: Intravascular Hemolysis,
o tumor markers are substances that Hemoglobinuria
are detected in cancer
Β2-microglobulin
α-1 glycoprotein (AAG) - found on the surface of nucleated cells
- aka OROSOMUCOID - present in high concentration on lymphocytes
- only protein that is negatively charged even in - needed in the production of CD8 cells for
acidic state due to salicylic acid present as a immunity
component of the protein o CD8 is found in the surface of every
Clinical Significance: nucleated cells
- Acute phase reactant
- increased levels: stress, carcinoma, Transferrin (Siderophilin)
rheumatoid arthritis(RA), inflammation, AMI, - major component of beta2 region
pregnancy, pneumonia, surgery - transport protein of iron
- prevents loss of iron through the kidney
α-2 macroglobulin Clinical Significance:
- largest major non-immunoglobulin - negative acute phase reactant
- inactivate proteases - increased levels: Iron Deficiency Anemia
- 800 kilo Dalton
Clinical Significance: Complement
- increased levels: nephrotic syndrome - participates in immune reaction
o NS – protein are filtered by kidney - linked to inflammatory response
and detected in urine; glomerular
problem Hemopexin
o highest in NS - binds w heme released from hgb → destroyed
o equal or higher than albumin in the liver → preserve body’s iron
concentration Clinical Significance:
NOTES: - acute phase reactant
- proteins are not normally present in urine - decreased levels: Hemolytic Anemia
since it is too big
Fibrinogen
Lipoprotein - one of the largest plasma proteins
- transports cholesterol, triglycerides and - use in clotting
phospholipids - thrombin cleaves fibrinogen to form fibrin
- most abundant coagulation factor which
Ceruloplasmin forms the fibrin clot
- transport protein for 90% of copper (other - not detected in the serum since it has been
10% is bound to albumin) used up to clot the blood
- copper containing protein

Annie June Audan

Clinical Significance: Total Protein Abnormalities
- acute phase reactant ▪ Hypoproteinemia
- Increase plasma volume (dilutional effect)
C-Reactive Protein (CRP) - An actual disease
- named because it binds with the C Protein loss through
Nephrotic syndrome
polysaccharide of the pneumococcus urine
- “general scavenger” Protein loss through
Clinical Significance: extrusion of plasma
- highly sensitive acute phase reactant Extensive burns (leaking of protein
- first inflammatory marker to appear through interstitial
- rapid presumptive test of bacterial vs viral fluids)
infection Hepatocellular disease /
Protein not synthesized
- increased levels: inflammatory diseases cirrhosis
- Highly sensitive – can detect inflammation
even in small amount ▪ Hyperproteinemia
- Non-specific – it cannot detect specific cause - not an actual disease
and area of the inflammation - not an overproduction of protein
- acute rheumatic fever, acute myocardial TP ALB GLOB DISEASE
infarction, RA, gout ↑ ↑ ↑ Dehydration
o ESR – more sensitive than CRP ↑ N ↑A Multiple myeloma;
Polyclonal and monoclonal
Immunoglobulins gammopathies
- Antibodies (Ab) ↓ ↓ ↓ Hyperaldoseteronemia/Salt
- synthesized by plasma cells Retention Syndrome
- for immune response ↓ ↓ N Malabsorption, inadequate
- IgG, IgA, IgM, IgE, IgD diet; Nephrotic syndrome
Clinical Significance: ↓ N ↓ Immunodeficiency
- increased levels: multiple myeloma, infections, syndromes
allergic reactions, hepatic dse
↓/N ↓ ↑/N Hepatic damage such as
cirrhosis, hepatitis and
Myoglobin
obstructive jaundice; burns,
- primary oxygen-carrying protein in striated trauma
skeletal and cardiac muscle
- hgb of the muscle
Specimen Considerations
- nephrotoxic
▪ Specimen: Serum is preferred; 24-hr urine and
Clinical Significance:
serous fluid can be used
- Cardiac marker – 1st to increase in AMI
▪ Hemolyzed and lipemic samples are not accepted
o Not really used due to its short life
span
Total Protein Measurement
- increased levels: AMI, muscular diseases
▪ Kjeldahl Method (reference method)
- Indirect method since it measures the nitrogen
Troponin
content
- Troponin I, Troponin T, Troponin C
- Average Nitrogen Content: 16%
Clinical Significance:
- 3 step method
- Gold standard marker for acute myocardial
(1) precipitation by trichloroacetic acid (TCA)
infarction
or tungstic acid
(2) acid digestion by H2SO4 + heat + cupric
Brain Natriuretic Peptide (BNP)
sulfate (as catalyst) = nitrogen is
- released in response to volume expansion and converted to ammonium ions
the increased wall stress of cardiac myocytes (3) quantitation by back titration with HCl or
- cardiac marker Nesslerization with HgI2 or KI

GC globulin
Nessler’s Gum ghatti Yellow
- Group specific component
reaction
- migrates between alpha 1 and 2 region
Berthelot’s Hypochlorite Blue
(belongs to neither group)
reaction
- exhibits affinity to vitamin D
▪ Biuret Method (routine method)
- cupric ions complex with peptide bond =
formation of violet colored chelate
- read at 540nm
- requires at least 2 peptide bonds to be
positive

Annie June Audan

Reagents: ▪ Smaller particles and more negatively charged
- Rochelle salt (sodium potassium tartrate) particles will migrate farther towards the anode
- NaOH
- Alkaline copper sulfate
- Potassium iodide
Electrophoretic Patterns
▪ Dye Binding
- based on the ability of most proteins to bind
dyes (read at 465nm – 595nm)
- dyes: Bromphenol Blue, Ponceau S, Amido
Black, Limassine Green, Coomassie Brilliant
Blue

▪ Lowry/Folin Ciocaltau (highest analytical sensitivity)

- Oxidation of phenolic compounds to a DEEP
BLUE color

▪ Refractometry
- rapid and simple test
- measures refractive index of solutes in serum

Albumin Measurement
▪ Salt Fractionation
- Globulins are precipitated in high salt
concentrations (ammonium sulfate)
- Albumin is quantified using biuret reaction
- Reference range:
Total Protein = 6.5-8.3 g/dL
Albumin = 3.5-5.5 g/dL
- Conversion factor for both (g/dL to g/L) = 10
Normal serum protein electrophoretic pattern
▪ Dye Binding
- Albumin binds to dye causing shift in
absorption maximum

Methyl orange Non specific for albumin

HABA Many interferences such
2-Hydroxyazobenzen- as salicylates & bilirubin
4'-Carboxylic Acid
Bromcresol green Most commonly used
Bromcresol purple Most specific, sensitive
and precise
Beta-Gamma Bridging: Liver Cirrhosis
Protein Electrophoresis
▪ Proteins are negatively charged at pH 8.6 (anion)
and they move towards the anode
▪ After electrophoresis → fixative →stained →
densitometry (measures the absorbance of stain –
concentration of the dye and protein band)

Components
1. Support Media
o Agarose- most commonly used for
lipoproteins
o Cellulose acetate- most commonly used
for SPE
o Polyacrilamide- most commonly used for
isoenzymes Monoclonal Spike: Monoclonal Gammopathy
o Nitrocellulose- most commonly used for (Multiple Myeloma and Waldenstrom’s
western blot Macroglobulinemia)
2. Buffer- maintain a constant pH (pH 8.6)
3. Electric current

▪ Proteins are (-) charged, so sample is placed on the

negative side

Annie June Audan

 decreased α-1 Antitrypsin: Emphysema

decreased Albumin and increased α-2 region:

Nephrotic Syndrome

Increased α1 and α2, slightly decreased albumin: acute

inflammation

Annie June Audan

 ACTIVITY 6: PROTEIN DETERMINATION

Specimen Consideration
✓ Serum is preferred; 24-hr urine and serous
fluid can also be used.
o Pleural
o Peritoneal
o Pericardial
✓ Lipemic and hemolyzed samples should not
be used.

A. Electrophoresis
✓ Migration of charged particles in an electric
field. C. Electrophoretic Patterns
✓ Separates proteins on the basis of their
electric charge densities. Beta-Gamma Bridging: Liver Cirrhosis
✓ During electrophoresis, proteins are negatively This is seen in patients with hepatic cirrhosis.
charged at pH 8.6 (anion) and they move
towards the anode
✓ PROTEINS ARE NEGATIVELY CHARGED
✓ After electrophoresis → fixative → stained →
densitometry
✓ Supporting Media for Electrophoresis:
1. Paper electrophoresis
2. Starch gel - separates by surface charge
and molecular size
3. Cellulose acetate – separates by
molecular size; most common for SPE
4. Agarose gel – neutral; separates by
electrical charge
5. Polyacrylamide gel – neutral; separates
by charge and molecular size; separates Monoclonal Spike: Monoclonal Gammopathy
proteins into 20 fractions; used to study (Multiple Myeloma and Waldenstrom’s
isoenzymes. Macroglobulinemia)
This is seen in cases of monoclonal gammopathy
B. Densitometry (multiple myeloma and Waldenstroms disease)
✓ measures the absorbance of stain
✓ concentration of the dye and protein band
✓ scan and quantitate electrophoretic pattern.

Increased α1 and α2, slightly decreased albumin:

acute inflammation
Increased alpha 1 and 2, slightly decreased albumin

Annie June Audan

 Chronic inflammation 1st Step precipitation by trichloroacetic acid (TCA)
Increased alpha 1 and 2 and gamma region, slightly or tungstic acid
decreased albumin 2nd Step acid digestion by H2SO4 + heat + cupric
sulfate (as catalyst) = nitrogen is
converted to ammonium ions
3rd Step quantitation by back titration with HCl or
Nesslerization with HgI2 or KI = yellow to
yellow-orange (+)

II. Biuret Method

• most widely used; routine method
• cupric ions complex with peptide bond =
formation of violet colored chelate (+) read at
540 nm
• requires atleast 2 peptide bonds to be positive
Nephrotic Syndrome • reagents:
decreased Albumin and increased α-2 region: o Rochelle salt (sodium potassium
Nephrotic Syndrome tartrate)
o Alkaline copper sulfate
o NaOH
o Potassium iodide

III. Dye Binding

• For total protein and albumin
• based on the ability of most proteins to bind
dyes (read at 465nm – 595nm)
• dyes:
o bromphenol blue
o ponceau S
o amido black
o limassine green
decreased α-1 Antitrypsin: Emphysema o coomassie brilliant blue
Decreased alpha-1 antitrypsin o Bromcresol purple – most sensitive,
specific and precise
o Bromcresol green – most commonly
used

IV. Salt Fractionation

• For Total protein
• globulins are precipitated in high salt conc
(ammonium sulfate)
• albumin is quantified using biuret reaction
• reference range:
o TP = 6.5-8.3 g/dL
o Alb = 3.5-5.5 g/dL
• Conversion factor for both (g/dL to g/L) = 10

D. Other Methodologies

Total Protein Methods Albumin Methods

1. Kjeldahl Method 1. Salt Fractionation
2. Biuret 2. Dye Binding
3. Dye Binding

I. Kjeldahl Method
• reference method; measures the nitrogen
content
• assumes average nitrogen content of 16%
(actual nitrogen content is 15.1% to 16.8%)
• assume no protein of significant concentration
are lost in the precipitation step.

Annie June Audan

 glutamine in CSF and may be fatal if ammonia
NON-PROTEIN NITROGEN SUBSTANCES
levels remain high.
o 100% survival if ammonia is 5x below
- Non-Protein Nitrogen Substances (NPNs) are normal
nitrogen-containing compounds that are not - Hepatic encephalopathy
proteins or polypeptides. - Specimen Consideration:
- They are waste products removed from the o Serum is NOT used;
blood by kidneys. o EDTA/heparinized plasma is
- NPNs are initially and primarily used to assess recommended
kidney function but not all are specific for - Specimens for ammonia level determination
kidney as some of them can assess the liver must be placed on ice immediately and
and/or muscle diseases. analyzed ASAP.
- NPNs does not have protein, nitrogenous o Since Whole Blood ammonia
substances. concentration increases rapidly due to
in vitro amino acid deamination
Clinically Approximate Approximate - RBC contains 2-3 times as much ammonia as
Significant plasma conc urine conc plasma
NPNs (% of total (% of total o So ammonia should be processed
NPN) NPN) immediately
Urea 45-50 86 - Samples should be centrifuged at 0-4°C within
Amino Acids 25 - 20 minutes of collection
Uric Acid 10 1.7
Creatinine 5 4.5 METHODS FOR AMMONIA DETERMINATION
Creatine 1-2 - 1. Conway Method
Ammonia 0.2 2.8 - Ammonia is released from alkaline solution and
determined by back titration with acid

Ammonia 2. Ion-Selective Electrode

- Ammonia (NH3) is a product of amino acid - Change in pH of solution as ammonia diffuses
deamination during protein metabolism through semi-permeable membrane =
- It is present in very small concentration in the measured potentiometrically
blood in normal individuals
- It is a neurotoxic substance and could, 3. Spectrophotometry
therefore, damage the brain and the peripheral - Ammonia + bromphenol blue = (+) BLUE dye
nervous system if found in high concentrations
- Metabolism and source of ammonia: 4. Enzymatic Method (indirect method)
- Rate of disappearance of NADH read at 340
nm
- Coupled Enzyme Reaction:
a. Urease
Urea + 2 H2O →ammonia + ammonium carbonate
b. Glutamate Dehydrogenase
Ammonia + oxaloglutarate + NADH + H →
glutamate + H2O + NAD+

Urea
- Blood Urea Nitrogen (BUN) is the major
excretory product of protein metabolism. It is
the NPN found in highest concentration in
- 85% of ammonia goes to the liver so it can be blood
synthesized into urea. - Highest concentration of NPN found in the
- Ammonia can be used to assess liver function blood
since it is consumed by parenchymal cells of - Metabolism and source of urea:
liver to be converted to urea. It may be used to
evaluate impending hepatic coma and terminal
stages of hepatic cirrhosis
o If liver is damaged, ammonia will not
be converted into urea and will
concentrate in the blood, damaging
the brain and PNS.
- Reye’s Syndrome is an acute metabolic
disorder of the liver that can present with
severe fatty infiltration of liver that occurs
mostly in children. It causes increased - Urea cannot be formed without ammonia
ammonia levels in blood and increase (from breakdown of protein)

Annie June Audan

 - Ammonia is initially produced as a result of the - Whole blood must be deproteinized to
deamination of amino acids during protein eliminate hemoglobin
synthesis. Because ammonia is a toxic - BUN / CREATININE RATIO differentiates the
substance, it is converted to a lesser toxic cause of abnormal urea concentration (Normal
substance in the liver, thus urea is formed. ratio: 10:1 or 20:1)
- Urea is the first metabolite to increase during
kidney disease Pre-renal HIGH Increased
- After filtering their nitrogen, liver forms urea BUN/Crea BUN
molecules ratio Normal Crea
- Urea’s excess C, H, and O can be used to Renal LOW Increased
release energy BUN/Crea BUN
- Urea leaves the liver through the bloodstream ratio Increased
and travels to the kidneys. Crea
Post-renal HIGH Decreased
BUN (Blood Urea Nitrogen) BUN/Crea BUN
- One of the tests requested to assess the liver ratio Normal Crea

CONDITIONS ASSOCIATED WITH ABNORMAL BUN METHODS FOR AMMONIA DETERMINATION

LEVELS
Azotemia
- increased BUN without kidney disease
Uremia
- increased BUN with kidney disease
a. Pre-renal: reduced renal blood flow, high
CHON metabolism, high CHON diet
b. Renal: renal disease, renal failure
c. Post-renal: urinary tract obstruction
Decreased BUN
- low dietary CHON, severe liver disease
▪ Diacetyl Monoxime Method (direct method)
- colorimetric method that is the most
inexpensive but non-specific
Urea + diacetyl monoxime →diazine (yellow) =
Fearon’s Reaction
- addition of arsenic thiosemicarbazide = color
enhancer and exclude protein interference
▪ Isotope-Dilution Mass Spectrometry (direct method)
- Reference method
▪ Enzymatic Method (indirect method)
- Coupled Enzyme Reaction: (1) Urease (2)
Glutamate Dehydrogenase
Urea + 2 H2O → ammonia + ammonium carbonate
Ammonia + oxaloglutarate + NADH + H →glutamate +
H2O + NAD+
- rate of disappearance of NADH read at 340 nm

▪ Nesslerization Reaction (indirect method)

- Reagent: KI and HgI2
- Stabilizer: gum ghatti
Ammonia + Nessler’s rgt = (+) yellow to yellow-orange
compound

MEASURING UREA IS USED TO: ▪ Berthelot Reaction (indirect method)

- evaluate renal function - Reagent: Na hypochlorite
- assess hydration status - Stabilizer: sodium nitroprusside
- determine nitrogen balance Ammonia + phenol hypochlorite = (+) indophenol blue
- aid in the diagnosis of renal disease
- verify adequacy of dialysis ▪ Indicator Dye (indirect method)
- Previous methods measure nitrogen content in - ammonium + pH indicator = color change
a protein-free filtrate
▪ Conductimetry (indirect method)
- Assess the increase in conductivity rate due to
MEASURING UREA:
formation of electrically charged ammonium
- BUN x 2.14 = Urea concentration
ions from urea
- Urea is reported in mmol/L
- Specific and rapid
- conversion factor:
o mg/dL to mmol/L = 0.357
ACTIVITY 10-A: NPN DETERMINATION
- reference range (plasma):
BLOOD UREA NITROGEN
o 6-20 mg/dL (2.1-7.1 mmol/L)
- Specimen:
- Urea is a non-protein nitrogenous substance
o plasma
that is present in highest concentration in the
o serum
blood
o urine
- It is the major excretory product of protein
- Ammonia-containing anticoagulant, Na
metabolism
fluoride and Na citrate should not be used
- Most of the urea is excreted in the kidneys
- Use a non-hemolyzed sample
and/or through the GI and skin

Annie June Audan

 - Only 40% of urea is reabsorbed REAGENT COMPOSITION
- Historic assays for urea were based on Phosphate Buffer (pH
measurement of nitrogen, thus the term, Blood 7.0)
Urea Nitrogen (BUN) Reagent 1 Sodium salicylate
- Only the nitrogen part of urea is quantitated Sodium nitroprusside
since it reflects the actual concentration of the EDTA
entire urea compound Phosphate buffer (pH <13)
Reagent 2
Hypochlorite
BUN measurement Enzyme Concentration Urease
- Enzymatic methods are most commonly used
Urea: 80 mg/dL or
in the measurement of urea
13.3 mmol/L
Standard
Equivalent to BUN:
37.28 mg/dL or 6.2 mmol/L

PRINCIPLE:
Modified Berthelot’s reaction
- Ammonium ions react with hypochlorite and
salicylate to form a green dye
- Measured spectrophotometrically at 570-
600nm
ENZYMATIC METHODS - The increase in absorbance is proportional to
BUN concentration
Methods use a Enzymatic See Figure 12-
production of 2 PIPETTING SCHEME AND PROCEDURE
similar first
ammonium 1. 0.001 mL Patient’s Serum + 1.0 mL Reagent 1
step— 2. Mix and incubate for 5 minutes at 37°C
catalyzed by ion (NH4+) from 3. Add 1.0 mL Reagent 2
urease urea 4. Mix and incubate for 10 minutes at 37°C
5. Measure the absorbance of the standard and
Enzymatic Used on many
the sample against the reagent blank within
reaction of NH4+, automated
60 minutes
GLDH coupled 2-oxoglutarate instruments;
enzymatic and NADH to best as a CALCULATION OF UREA AND BUN
form glutamate kinetic CONCENTRATION
and NAD+ measurement UREA
NH4+ + pH Used in SERUM AND
mg/dL mmol/L
indicator → color automated PLASMA
change systems, 𝑠𝑎𝑚𝑝𝑙𝑒
x
multilayer 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 80 13.3
Indicator dye
film reagents, URINE mg/dL Mmol/L
and dry 𝑠𝑎𝑚𝑝𝑙𝑒
x
reagent strips
𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 80.8 1343
Conversion of Specific and
unionized urea rapid BUN
to NH4+ and
SERUM AND
Conductimetric mg/dL mmol/L
CO32– results in PLASMA
𝑠𝑎𝑚𝑝𝑙𝑒
increased x
conductivity
𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 37.28 6.2

OTHER METHODS URINE mg/dL Mmol/L

𝑠𝑎𝑚𝑝𝑙𝑒
Detection of Proposed x
𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 37065 626.2
characteristic reference
fragments method CONVERSION FACTOR
following • BUN Concentration = 0.466 x Urea Concentration
Isotope dilution
mass ionization; • Urea Concentration = 2.14 x BUN Concentration
quantification
spectrometry using
isotopically
labeled
compound

Annie June Audan


Uric Acid
- product of catabolism of purine nucleic acids (in
the liver)
- 70% of blood uric acid is chiefly excreted by the
kidneys; 98-100% is reabsorbed
- Nearly all UA in plasma is present as
monosodium urates
- Increased levels of uric acid is toxic to the
kidneys (nephrotoxic)
- Relatively insoluble to the plasma
CONDITIONS ASSOCIATED WITH ABNORMAL UA
LEVELS
▪ Gout
- Defect in Uric Acid metabolism
- May be due to overproduction of Uric Acid
- Pain & inflammation of joints by precipitation of
sodium urates in tissues and/or joints
- Increased UA in the blood
▪ Chronic Alcoholism
- Alcohol inhibits uric acid excretion, thereby
increasing UA in the blood
▪ Lesch-Nayhan Syndrome
- X-linked
- caused by complete deficiency of hypoxanthine
guanine phosphoribosyltransferase (HGPRT)
that causes an overproduction of uric acid in
the body
- cause orange sand urine in diaper
▪ Renal Disease & Purine-rich Diet
▪ Increased Metabolism of Cell Nuclei
- seen in multiple myeloma, leukemia, lymphoma
X-linked
▪ Liver Disease and Fanconi’s Syndrome cause
decreased levels of Uric Acid
▪ Measuring UA is used to:
- assess inherited disorders of purine
metabolism;
- to confirm diagnosis and monitor treatment of
gout;
- to assist in the diagnosis of renal calculi;
- to prevent uric acid nephropathy during
chemotherapeutic treatment;
- and to detect kidney dysfunction
▪ Specimen:
o plasma,
o serum,
o urine
▪ Measuring UA:
- conversion factor: mg/dL to μmol/L = 0.0596
- reference range: 3.5 - 7.2 mg/dL (males)
2.6 - 6.0 mg/dL (females)
▪ hemolyzed, icteric, lipemic must be avoided
▪ salicylates and thiazides increase uric acid levels
▪ UA is stable after separation of RBC from serum or
plasma
▪ addition of thymol increases stability to bacterial
destruction
METHODS FOR URIC ACID DETERMINATION
▪ Isotope-Dilution Mass Spectrometry (direct method)
- Reference method
▪ Direct Redox Methods (oldest method)
- Uric Acid is readily oxidized to allantoin
- Alkalinizing agent – to inactivate non-uric acid
reactants
*Sodium Cyanide (NaCN): Folin, Brown, Newton,
Benedict
*Sodium Carbonate (Na2CO3): Caraway
▪ Caraway Method
- (+) tungsten blue read at 650-700nm
Uric acid + Na2CO3 + tungsten blue + phosphotungstic
acid = allantoin +CO2
Enzymatic Methods
▪ Uricase Method/Blaunch and Kock Method
▪ Caraway Method
Initial rxn: Uric acid + O2 + 2 H2O
→ allantoin + CO2 + H2O2
(coupled enzymatic method) = initial reaction + either:
Catalase OR Peroxidase + H2O2 + indicator dye = (+)
change of color
Creatinine
▪ end product of muscle metabolism
▪ LIVER: arginine, glycine, methionine →Creatine
→Creatinine
▪ MUSCLES: creatine → Phosphocreatine
→Creatinine
▪ Very efficient test to evaluate renal function because:
- it is removed by glomerular filtration, therefore,
plasma creatinine assess glomerular filtration
- Small amounts are reabsorbed
- Unaffected by other factors
▪ Plasma creatinine levels usually normal, but urinary
creatinine is elevated
▪ It is also not elevated unless 25-50% of the nephrons
are destroyed
CONDITIONS ASSOCIATED WITH ABNORMAL CREA
LEVELS
▪ Muscular Dystrophy
▪ Kidney Disease
▪ Measuring creatinine is used to:
- assess sufficiency of kidney function
- assess severity of kidney damage
- monitor the progression of kidney disease.
▪ Specimen: plasma, serum, urine may be used
▪ Measuring creatinine:
- conversion factor: mg/dL to μmol/L = 88.4
- reference range:
o (M) 0.9 - 1.3 mg/dL or 80 - 115 μmol/L
o (F) 0.6 – 1.1 mg/dL or 53 – 97 μmol/L
▪ hemolyzed, icteric, lipemic must be avoided
▪ No strenuous exercise; no fist clenching
Annie June Audan
 METHODS FOR CREATININE DETERMINATION - Creatine is synthesized primarily in the liver
▪ Isotope-Dilution Mass Spectrometry (direct method) from arginine, glycine, and methionine. It is
- Reference method then transported to other tissues, such as
muscle, wherein it will serve as a high energy
▪ Jaffe Method source.
- Reagent: alkaline picrate solution (freshly - Creatine phosphate loses phosphoric acid and
prepared) creatine loses water to form the cyclic
compound, creatinine, which diffuses into the
Creatinine + jaffe rgt = RED-ORANGE (Janovsky plasma
complex) - About 99% is excreted in the urine
- Plasma creatinine is inversely related to
▪ Modified Jaffe Method glomerular filtration rate (GFR)
- With the addition of an adsorbent - It is commonly used to assess renal filtration
o Fuller’s Earth reagent: aluminum function
magnesium silicate
o Lloyd’s reagent: sodium aluminum silicate CREATINE MEASUREMENT
▪ Modified Jaffe Method
- With the addition of an adsorbent
CHEMICAL METHODS BASED ON JAFFE REACTION
Jaffe reaction In alkaline
Enzymatic Methods
solution,
▪ Creatininase-CK Method
creatinine +
- measure change in NADH to NAD+
picrate → red-
orange complex
Jaffe-kinetic Jaffe reaction Positive bias
performed from α-keto
directly on acids and
sample; detection cephalosporins;
of color formation requires
timed to avoid automated
interference of equipment for
non-creatinine precision
▪ Creatininase-H2O2 Method chromogens
- measure formation of color as Jaffe with Creatinine in Adsorbent
benzoquinoneimine dye forms adsorbent protein-free improves
filtrate adsorbed specificity;
onto Fuller’s previously
earth (aluminum considered
magnesium reference
silicate), then method
reacted with
alkaline picrate to
form colored
complex
Jaffe without Creatinine in Positive bias
adsorbent protein-free from ascorbic
filtrate reacts acid, glucose,
ACTIVITY 10-B: NPN DETERMINATION with glutathione, α-
CREATININE alkaline picrate to keto acids, uric
- Creatinine is a product of muscle metabolism form colored acid, and
- It is formed from the interaction between
complex cephalosporins
creatine and ATP which is converted to
ENZYMATIC METHODS
creatine phosphate and ADP with the help of
Creatininase- In a series of Adapted for
the enzyme, creatine kinase.
H2O2 enzymatically use as dry slide
catalyzed method;
reactions, potential to
creatinine is replace Jaffe;
hydrolyzed to no interference
creatine, which is from
converted to acetoacetate or
sarcosine and cephalosporins;
urea. Sarcosine is some positive
oxidized to bias due to
glycine, CH2O, lidocaine
and H2O2.
Peroxidase-
catalyzed

Annie June Audan

 oxidation of a 24-HOUR URINE
colorless Conc = mg/dL x mL Conc = mg/24h x
substrate 0.00884
urine/24 hr x 0.01
produces a
(mmol/24h)
colored product + (mg/24hr)
H2O
Creatininase- In a series of Lacks
CK reactions sensitivity; not Conversion Factor
catalyzed by the used widely • mg/dL x 88.402 = μmol/L
enzymes • μmol/L x 0.0113 = mg/dL
creatininase,
creatine kinase, ACTIVITY 10-C: NPN DETERMINATION
pyruvate kinase, SERUM URIC ACID
and lactate - Uric acid is the product of catabolism of the
dehydrogenase, purine nucleic acids
NAD+ is - Although it is filtered by the glomerulus and
produced and secreted by the distal tubules into the urine,
measured as most uric acid is reabsorbed in the proximal
a decrease in tubules and only <1% is excreted
absorbance - Uric acid is relatively insoluble in plasma and,
at high
OTHER METHODS
- concentrations, can be deposited in the joints
Isotope Detection of Highly specific;
and tissue, causing painful inflammation
dilution mass characteristic accepted
- Although uric acid measurement may assess
spectrometry fragments reference
kidney function, uric acid is not as reliable as
following method
urea and creatinine
ionization;
quantification
SERUM URIC ACID MEASUREMENT
using isotopically
CHEMICAL METHODS
labeled
compound Phosphotungstic In carbonate Nonspecific;
acid solution requires
REAGENT COMPOSITION (Na2CO3/OH–), protein
Reagent 1 Picric Acid
uric acid + removal
Sodium Hydroxide
H3PW12O40 +
Sodium nitroprusside O2 →allantoin
Standard Creatinine: + tungsten
2 mg/dL or 176.8 μmol/L blue + CO2
ENZYMATIC METHODS

PRINCIPLE: Similar first step— Enzymatic See Figure

Jaffe reaction Catalyzed production of 12-3
- forms in alkaline solution an orange-red allantoin
Very
colored complex with picric acid
from uric acid specific
Creatinine + Picric Acid Creatinine-picrate complex
- measured spectrophotometrically at 520nm Coupled H2O2 + Readily
- the increase in absorbance is proportional to enzymatic— indicator dye automated;
creatinine concentration Peroxidase → colored reducing
compound agents
Pipetting Scheme and Procedure
interfere
1. 0.01 mL Patient’s Serum + 1.0 mL Reagent 1
2. Mix. After 30 seconds, read A1. Spectrophotometric Decrease in Hemoglobin
3. After 2 minutes, read A2. absorbance at and
4. A2-A1 = ΔA xanthine
293 nm
measured interfere
OTHER METHODS
CALCULATION OF CREATININE CONCENTRATION
SERUM OR PLASMA Isotope dilution Detection of Proposed
𝛥𝐴 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔/𝑑𝐿) 𝛥𝐴 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔/𝑑𝐿) mass characteristic reference
Conc = 2.0 x Conc = 176.8x
𝛥𝐴 𝑆𝑇𝐷 𝛥𝐴 𝑆𝑇𝐷 fragments method
following
ionization;
URINE quantification
𝛥𝐴 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔/𝑑𝐿)
Conc = 10 x using
𝛥𝐴 𝑆𝑇𝐷
isotopically

Annie June Audan

 labeled
compound

REAGENT COMPOSITION
Reagent 1 Phospate buffer (pH 7.0)
4-aminophenazone
DCHBS
Uricase
Peroxidase
Standard Uric Acid:
8 mg/dL or 476 μmol/L

Principle
Uricase method
- uric acid is converted to allantoin and
hydrogen peroxide in the presence of uricase
- H2O2 reacts with 3,5-dichloro-2
hydroxybenzenesulfonic acid (DCHBS) and 4-
aminophenazone (PAP) to give a red-violet
- quinoneimine dye
Uric Acid + O2 + 2 H2O2 -----uricase→allantoin + CO2
+ H2O2
2 H2O2 + DCHBS + PAP -----
peroxidase→quinonemine + HCl + 4 H2O
- measured spectrophotometrically at 520nm
- the increase in absorbance is proportional to
SUA concentration

Pipetting Scheme and Procedure

1. 0.02 mL Patient’s Serum + 1.0 mL Reagent 1
2. Mix and incubate for 10 minutes at 37°C
3. Measure the absorbance of standard and the
sample against reagent blank within 15
minutes

CALCULATION OF SUA CONCENTRATION

SERUM OR PLASMA
𝐴𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔/𝑑𝐿)
𝑐𝑜𝑛𝑐 = 8 𝑥
𝐴𝑏𝑠 𝑆𝑇𝐷
𝐴𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 (μmol/L)
𝑐𝑜𝑛𝑐 = 476 𝑥
𝐴𝑏𝑠 𝑆𝑇𝐷
URINE
ΔA 𝑠𝑎𝑚𝑝𝑙𝑒 (mg/dL)
𝑐𝑜𝑛𝑐 = 88 𝑥
ΔA 𝑆𝑇𝐷
ΔA 𝑠𝑎𝑚𝑝𝑙𝑒 (μmol/L)
𝑐𝑜𝑛𝑐 = 5235 𝑥
ΔA 𝑆𝑇𝐷

Annie June Audan


LIVER FUNCTION
MACROSCOPIC ANATOMY OF THE LIVER
- The liver is a very large and complex organ
that weighs 1.2 to 1.5kg in a healthy adult
- It is located beneath and attached to the
diaphragm & protected by the lower rib cage
- It is divided unequally into 2 lobes, the right
lobe (bigger) and the left lobe (smaller)
- The liver has two sources of blood supply:
hepatic artery (supplies oxygen-rich blood)
and portal vein (supplies nutrient-rich blood)
MICROSCOPIC ANATOMY OF THE LIVER
- The functional unit of liver is called lobules
which are responsible for the metabolic and
excretory functions
- The portal triad present in each lobule consists
of a hepatic artery, portal vein and bile duct
- The liver contains two cell types:
- Hepatocytes- perform major functions of the
liver
- Kupffer cells- macrophages
BIOLOGICAL FUNCTIONS OF THE LIVER
Excretory and Secretory
- processing and excreting endogenous and
exogenous substances into bile or urine
- Bilirubin: major heme waste product (liver is
the only organ capable of disposing bilirubin)
- Bile: contains bile acids, bile salts, bile
pigments, cholesterol and other blood
substances
- Bilirubin is the principal pigment in bile
BILIRUBIN METABOLISM
1. Bilirubin results from the breakdown of RBCs
2. As RBCs are broken down, heme is released.
3. Heme is converted to biliverdin by heme
oxygenase.
4. Biliverdin is converted to unconjugated
bilirubin (B1) by biliverdin reductase.
5. B1 is insoluble to water and could not freely
pass through the blood, therefore, it attaches
to albumin so that it can travel going to the
liver.
6. Albumin detaches as B1 enters the liver. In the
liver, B1 is converted to conjugated bilirubin
(B2) by UDPGT.
7. B2 is then released in the bile duct going to
the intestines.
8. In the intestines, intestinal bacteria converts
B2 into mesobilirubinogen → mesobilirubin
→urobilinogen (colorless)
9. Urobilinogen is oxidized into urobilin (orange)
10. 80% of urobilin → stercobilinogen →
stercobilin (color pigment in feces)
11. Majority of the remaining 20% of urobilin will
be absorbed by the hepatic circulation to be
recycled in the liver and re-excreted. The
minority will enter systemic circulation, filtered
by the kidneys, and excreted in the urine.
Annie June Audan
 Metabolism
- The liver metabolizes many biochemical DISEASE/DISORDER TB B1 B2
products such as: Gilbert Bilirubin
1. Carbohydrates disease transport deficit
↑ ↑ N
- use of glucose for its own cellular energy Deficiency of
- circulate glucose in peripheral tissues UDPGT
- storage of glucose as glycogen Criggler- Conjugation
2. Lipids Najjar deficit
- lipids, lipoproteins, cholesterol and fatty acids Syndrome Type 1:
3. Proteins complete
- almost all proteins are synthesized by the liver absence of
except immunoglobulins and adult hgb ↑ ↑ ↓
conjugation
- processing and excreting endogenous and Type 2: severe
exogenous substances into bile or urine deficiency
Prone to
Detoxification kernicterus
- Liver serves as a barrier to prevent toxic and Dubin- Bile excretion
harmful substances from reaching the Johnson deficit
systemic circulation Defective
- First pass: every substance absorbed must removal of
first pass the liver bilirubin from
Mechanism of detoxification in the body: it ↑ N ↑
- liver cells
binds to the substance to either inactivate the Appearance of
compound or chemically modify it so it can be dark-stained
excreted granules in liver
- Cytochrome P-450 isoenzymes: helps in the biopsy
detoxification of drugs that take place in the Rotor Clinically similar
liver microsomes Syndrome to Dubin-
Johnson but
LIVER FUNCTION ALTERATIONS DURING DISEASE ↑ N ↑
without dark-
I. Jaundice stained
- from the French word jaune meaning yellow granules
- refers to the yellowish discoloration of the Physiologic Jaundice of the
skin, eyes and mucus membranes most often jaundice newborn
resulting from the accumulation or retention of because of ↑ ↑ N
bilirubin underdeveloped
- normal value: 1.0-1.5 mg/dL liver
- jaundice is not noticeable to the naked eye Lucey- Presence of
(overt jaundice) until bilirubin levels reach 3.0- Discroll circulating ↑ N ↓
5.0 mg/dL Syndrome inhibitor of B2
- icterus: yellow discoloration of serum or
plasma sample due to high levels of bilirubin
c. Post-Hepatic jaundice
Jaundice based on Site of Disorder - the problem causing jaundice occurs after the
a. Pre-hepatic jaundice conjugation of bilirubin in the liver
- aka unconjugated hyperbilirubinemia - most often caused by biliary obstructive
- the problem causing jaundice occurs before disease such as gallstones or tumors that
liver metabolism block the flow of conjugated bilirubin into the
- increased levels of bilirubin being presented to bile canaliculi
the liver such as hemolytic anemia - stool becomes clay-colored and urine
- Hemolytic anemia results from increased RBC becomes dark
destrunction, thus, releasing increased
amounts of RBC degradation products such II. Cirrhosis
as bilirubin - scar tissue replaces normal, healthy liver
- processing and excreting endogenous and tissue, blocking the blood flow and preventing
exogenous substances into bile or urine the normal function of the liver
- the liver increases its function to keep up with - signs & symptoms: fatigue, nausea,
the increased levels of bilirubin to be unintendedweight loss, jaundice, GI bleeding,
processed generalized itching and swelling of the legs
- causes an increase in B1 and abdomen
o properties of B1: water insoluble, - has poor prognosis; considered irreversible,
bound to albumin, not filtered by the but can delay further progression of the
kidneys, not present in urine disorder

b. Hepatic jaundice
- the problem causing jaundice occurs in the
liver itself (intrinsic liver defect or disease)

Annie June Audan

III. Tumors b. Hepatitis B
- Primary Liver Cancer: begins in the liver cells - aka serum hepatitis or long-incubation
- Metastatic Liver Cancer: tumors from other hepatitis
parts of the body spread; more common - detectable in all body fluids
- Benign tumors: does not spread - transmission: parenteral, perinatal, sexual
- Ex. Heptocellular adenoma, Hemangioma
o Malignant tumors: spread from one
organ to another
- Ex. Heptocellular carcinoma (HCC)
o Most common malignant tumor of the
liver

IV. Reye’s Syndrome

- acute illness characterized by non-
inflammatory encephalopathy and fatty
degeneration of the liver
- almost exclusively found in children
- most often caused by a viral disease that
subsequently developed into Reye’s Syndrome
- the encephalopathy is characterized by a
progression from mild confusion (stage 1)
through progressive loss of neurologic
function to loss of brain stem reflexes (stage
5)
- characterized by: mild hyperbilirubinemia,
three-fold increase in ammonia and
aminotransferases
V. Drug and Alcohol-Related Disorders
- Common mechanism of drug toxicity is via
immune-mediated injury to the hepatocytes
- Ethanol: most common drug associated with
hepatic toxicity; mild intake causes mild
toxicity to the liver but chronic, heavy
consumption may lead to alcoholic cirrhosis
- Alcohol dehydrogenase: enzyme that breaks
down alcohol, as the stomach and intestines
transport alcohol to the liver for metabolism
alcohol → acetaldehyde → acetate
- Prolonged heavy intake of alcohol may lead
to:
steatohepatitis→ hepatic fibrosis → hepatic
fibrosis
- Acetaminophen: most common drug
associated with serious hepatic injury
VI. Hepatitis
- presence of inflammation in the liver tissue
- causes: viral, bacterial, parasitic, radiation,
drugs, chemical autoimmune and toxins
o viral hepatitis: most common
Serological markers:
Hepatitis B Surface Antigen (HBsAg)
- aka Australian antigen and hepatitis-
associated antigen
- routine test performed on all blood donation
units
- first marker to increase in acute infection
(before the onset of symptoms)
Hepatitis B Core Antigen (HBcAg)
- not present in the plasma of patients and
blood donors
- present only in the nuclei of hepatocytes
during acute infection
- Anti-HBc develop prior to Anti-HBs
- IgM anti-HBc: marker for acute infection
- aka Australian antigen and hepatitis-
associated antigen
- routine test performed on all blood donation
units
- first marker to increase in acute infection
(before the onset of symptoms)
Hepatitis B Envelope Antigen (HBeAg)
- closely associated with the core antigen
- is detected in acute and chronic infection
- reflects degree of infectivity
- e Ag is only detected when surface Ag is also
present

a. Hepatitis A
- aka infectious hepatitis or short-incubation
hepatitis
- most common form of viral hepatitis
- transmission: fecal-oral route
- Serological markers:
o IgM anti-HAV (marker for acute
infection)
o IgG anti-HAV (marker for chronic
infection)
c. Hepatitis C
(+)IgG anti-HAV (-)IgM anti-HAV = past infection
- aka non-A non-B hepatitis
- transmission: parenteral, sexual, blood
transfusion

Annie June Audan

 d. Hepatitis D
- co-infection with Hepatitis B (cannot exist
independently, therefore, requires the
presence of Hepatitis B)
- transmission: parenteral, sexual
- end color: red
- reported in Ehrlich units
- specimen: fresh 2-hour urine specimen or
fresh feces
- kept cool and protect from light

e. Hepatitis E • Enzymes
- more severe infection that Hepatitis A - Injury to the liver releases enzymes into the
- may be zoonotic circulation
- transmission: oral-fecal route - aid in the differential diagnosis, as well as
identifying the cause (functional or mechanical
problem)
LIVER FUNCTION TESTS a. Aspartate Aminotransferase
• Bilirubin Determination (Van den Berg Reaction) - former name: serum glutamic oxaloacetic acid
- classic diazo reaction; a reaction of bilirubin (SGOT)
with a diazotized sulfanilic acid to form a - more useful in detecting hepatocellular
colored product damage
- requires an accelerator to detect
unconjugated bilirubin, thus, allowing the b. Alanine Aminotransferase
measurement of total bilirubin - more useful in detecting hepatobiliary damage
- reaction without the presence of the - helps in the differential diagnosis of
accelerator measures conjugated bilirubin only hepatobiliary disease from osteogenic bone
- Fractionation of Bilirubin: disease
Bilirubin→ diazotized sulfanilic acid → Azobilirubin (TB) - former name: serum glutamic pyruvic acid
Bilirubin →diazotized sulfanilic acid → Azobilirubin (B2) (SGPT)
Total Bilirubin – B2 = B1 - more useful in detecting hepatocellular
- delta bilirubin: conjugated bilirubin covalently damage
bound to albumin; third fraction of bilirubin - more specific than AST
(along with conjugated and unconjugated
bilirubin); seen in significant hepatic c. Alkaline Phosphatase
obstruction and is measured as part of - former name: serum glutamic pyruvic acid
conjugated bilirubin (SGPT)
- specimen: serum (preferred) or plasma
- fasting sample is preferred as the presence of d. 5-Nucleotidase
lipemia increase measured bilirubin - more useful in detecting hepatobiliary damage
concentration - useful in the differentiation of ALP increase
- avoid hemolyzed samples due to liver conditions since 5-N does not
- protect from light exposure have a bone source
- more sensitive than ALP in metastatic liver
a. Evelyn-Malloy Method disease
- accelerator: 50% methanol
- end color: Red-purple (measured at 560 nm) e. Gamma-glutamyl Transferase
- similar to 5-N in terms of differential
b. Jendrassik-Grof Method diagnostic significance
- accelerator: caffeine-benzoate/sodium- - marker for chronic alcoholism
acetate
- Buffer: Na acetate
- pH: alkaline tartrate BILIRUBIN MEASUREMENT
- addition of ascorbic acid destroys excess REAGENT COMPOSITION
diazo reagent Total Bilirubin Reagent Sulfanilic acid
- end color: blue (measured at 500 nm) Hydrochloric acid
- Advantages of Jendrassik-Grof over Evelyn- Caffeine (accelerator)
Malloy Sodium benzoate
o not sensitive to pH changes Total Nitrite Reagent Sodium nitrite
o not affected by hemoglobin upto 750 Direct Bilirubin Reagent Sulfanilic acid
mg/dl Hydrochloric acid
o minimal turbidity Direct Nitrite Reagent Sodium nitrite
o adequate optical density
o insensitive to a 50-fold variation in
PRINCIPLE
protein concentration
Jendrassik and Grof method
- direct bilirubin reacts with diazotized
• Urobilinogen in Urine and Feces
sulphanilic acid to form a red azo dye
- urobilinogen (colorless) end product of
- measured at 546nm
bilirubin metabolism that can be further
- indirect bilirubin will only react with DSA in
degraded into urobilin (orange)
the presence of an accelerator
- reacts with Ehrlich’s reagent: para-
dimethylaminobenzaldehyde (PDAB)

Annie June Audan

 - absorbance is directly proportional to the
bilirubin concentration in the sample

Sulphanilic acid + sodium nitrite DSA

Bilirubin + DSA DIRECT BILIRUBIN
Bilirubin + DSA + accelerator TOTAL BILIRUBIN
Total bilirubin – Direct bilirubin = INDIRECT BILIRUBIN

Pipetting Scheme and Procedure

For TOTAL bilirubin
1. 1.0 mL TBR + 1 drop TNR
2. mix and incubate for 5 minutes
3. add 0.1 mL Patient’s serum
4. mix and incubate at room temperature for 10-
30 minutes
5. measure the absorbance of the sample
against the sample blank ΔA546
For DIRECT bilirubin
1. 1.0mL DBR + 1 drop DNR
2. mix and incubate for 2 minutes
3. add 0.1 mL Patient’s serum
4. mix and incubate at room temperature at
exactly 5 minutes
5. measure the absorbance of the sample
against the sample blank ΔA546

Calculation of Bilirubin Concentration

Bilirubin concentration = ΔA546 x 13.0 (mg/dL)
Bilirubin concentration = ΔA546 x 17.1 (μmol/L)
ΔA = A of sample blank – A of sample

Annie June Audan

 TUMOR MARKERS
Tumor
- uncontrolled growth of cells that occurs in
solid tissues such as an organ, muscle, or
bone causing the formation of a solid mass
Cancer
- disease wherein there is uncontrolled growth
of cells
Neoplasia
- general term for accelerated growth of tissue
Benign tumor
- “non-cancerous”; stays as it is and doesn’t
change
Malignant tumor
- “cancerous”; spreads and invades other
tissues or organs

Carbohydrates Antigens
CA 15-3, CA 25-29 Breast cancer
CA 19-9 Pancreatic cancer
CA 125 Ovarian cancer

Oncofetal antigens: normally expressed during fetal

life;
when it reappears in adulthood, it could mean cancer
Alpha Fetoprotein In childhood:
(AFP) Abundant protein synthesized
by the
fetal liver
Increased:
neural tube defects such as
ancephaly and spina bifida
Decreased: Down’s syndrome/
Benign Malignant
Epithelial tissue Adenomas Carcinomas trisomy 21
Connective Sarcomas In adulthood:
tissue
AFP production increases after
• Most common cancer in women: breast cancer the
• Most common cancer in men: prostate cancer stimulus of liver diseases

Tumor Markers NonCA: Viral hepatitis and

- Substances found in body tissues or fluids cirrhosis
that help show the presence or type of cancer of the liver
- Produced either by the cancer cells itself or by
the body in response to the presence of cancer CA: also made by primary liver
- Detected in a solid tumor or in circulating
tumors such as heptacellular
tumor cells presentin body fluids
carcinoma
3 Ideal Characteristics of a Tumor Marker: Carcinoembryonic In childhood:
• Tumor specific
antigen (CEA) produced in gastrointestinal
• Absent in healthy individuals
tissue
• Readily detectable in body fluids
during fetal development
Tumor markers should be:
In adulthood:
• Highly sensitive
• Highly specific CEA was discovered in
• Correlate with the cancer stage or tumor mass malignant
• Correlate with the prognosis
• Have a reliable prediction value tumors of the GI and pancreas
Non CA: liver disease,
inflammatory

Annie June Audan

 bowel disease, pancreatitis,
heavy
smoking
CA: GI, breast, pancreatic or
lung
cancer cells

Protein Tumor Markers

Serum M-Protein Plasma cell cancer
diagnosis and
Serum Free light chains
therapeutic monitoring
Hematologic malignancy
and/or
B2-Microglobulin lymphoproliferative
disorders prognosis

Receptor Tumor Markers

Estrogen Receptor Breast cancer hormonal
Progesterone Receptor therapy indicator

Breast cancer
Her-2/neu Determine the type of
therapy
Epidermal growth factor Head, neck, ovarian and
receptor (EGRF) cervical CA prognosis

Enzyme Tumor Markers

ALP ACP LPS
NSA AMS GGT
CK 5’N TRYPSIN
PSA LDH

Endocrine Tumor Markers

HCG CALCITONIN ACTH
CATECHOLAMINES SEROTONIN 5-HIAA
ADH GASTRIN GLUCAGON
INSULIN PRL ANDROGENS
TSH GH PTH
C-PEPTIDE VMA HVA

Annie June Audan

 CARDIAC FUNCTION
- Cardiac Function is the ability of the heart to
meet the metabolic demands of the body
- The heart has four chambers and four valves
that keep the blood flowing in the right
direction
- Cardiac marker: test to assist cardiac arrest
- Layers of the heart
o Epicardium (outer),
o Myocardium (middle)- bundles of
fibers
o Endocardium (innermost)-most
susceptible to ischemia
- Heart pump- because the function is
contracting and relax the myocardium
- Systematic flow of both oxygenated and
deoxygenated blood
BLOOD FLOW
Superior and Inferior Vena cava – (deoxygenated
blood) Right Atrium— Tricuspid Valve— Right
Ventricle—Pulmonary Valve— Pulmonary Artery—
LUNGS
From Lungs— Pulmonary Veins— (Oxygenated
Blood)— Left Atrium— Mitral Valve— Left Ventricle –
Aortic Valve— AORTA
CARDIOVASCULAR DISEASE (CVD)
- are a group of disorders of the heart and
blood vessels. CVDs are the number one
cause of death globally;
- more people die annually from CVDs than
from any other cause.
- Cardiac Function- to generate sufficient
amount
- SVC/IVC= SVC- blood flowing from the upper
body will pass thru, IVC- from lower
- Deoxygenated blood will enter the heart to
replenish from the lungs
- Cell (dead)- the functioning cells will able to
compensate to those who have gone/ healthy
cells will now function
- Heart attacks (myocardial infarction) and
strokes are usually acute events and are
mainly caused by a blockage that prevents
blood from flowing to the heart or brain.
- Heart- when it is damage, it will not generate
ACUTE CORONARY SYNDROME (ACS)
- Angina (chest pain)- reversible, not limited to
chest only, neglectable
- Classic manifestation of Cardiac Ischemia
o Squeezing of the chest
o Heavy chest pressure
o Burning feeling
o Difficulty breathing
- Non-Classical symptoms
o Viselike pressure
o Nausea
o Shortness of breath
o Abdominal pain
- Sudden cardiac disorder that Varrien in stable
and unstable angina to Myocardial Infarction.
- Stable angina (chest pain) = reversible
changes
- Unstable angina = irreversible change result
Myocardial injury
- Myocardial Infarction = extensive tissue
necrosis
ACUTE MYOCARDIAL INFARCTION (AMI)
- Referred as HEART ATTACK
- Worst, extensive tissue necrosis
- Imbalance of oxygen supple in heart muscle
- Happens when there’s increase demand of
oxygen, but the oxygen is not enough
- Can lead to necrosis- cell death, can result to
interruption of blood supply
- Common cause: Atherosclerosis
- Signs and Symptoms of heart attack (AMI):
• Difficulty breathing
• Feeling sick
• Feeling light headed
• Cold sweats
• Angina
• Pain in other parts of the body
STROKE
- Signs and Symptoms of stroke (Ischemic
attack)-BRAIN:
• Sudden weakness
• Numbness
• Fainting/unconsciousness
• Severe headache with no known
cause
• Difficulty walking, loss of balance or
coordination
- Stroke = first warning sign of an underlying
disease
- The normal artery wall has three layers (from
inside out):
o Intima
o media
o adventitia
Annie June Audan
 ATHEROSCLEROSIS
- Atherosclerosis is the narrowing or hardening
of the arteries due to plaque, major cause of
acute myocardial infarction
- Chronic Inflammatory disease
- Presence of plaque formation or atheroma
- Progressive accumulation of lipids, small
debris, collagen, smooth muscle cells,
macrophages, and connective tissues within
the intima of large and medium sized arteries
o Causes luminal narrowing and
decreased perfusion
- If plaque is rupture, it can cause blot clot-
completely impeded the blood flow

CARDIOVASCULAR DISEASES
PROCESS OF PLAQUE FORMATION: 1. Coronary Heart Disease (CHD)
1. Increased LDL will deposit in the tunica media - Disease of the blood vessels supplying the
(middle layer of artery wall) and becomes heart muscle
oxidized LDL (toxic to endothelial cells) - Impeded/ poor blood flow to the heart
2. Oxidized LDL activates endothelial cells and because of plaque formation
will express receptors for WBCs - Manifests as
(inflammatory response) o myocardial infarction
3. WBCs will bind to the receptors and will o angina
adhere to the endothelium due to activated o heart failure
endothelial cells and move to the tunica intima o sudden cardiac death
4. At the tunica intima, monocytes proliferate to
become macrophages. Macrophages take in 2. Cerebrovascular Disease (CBD= brain)
oxidized LDL and become foam cells - Disease of the blood vessels supplying the
(macrophage- will take up or engulf the brain
oxidized LDL forming foam cell) - Manifests as
5. Foam cells promote migration of Smooth o stroke
Muscle Cells (SMC) from tunica media to o transient ischemic attacks (short
intima. Increased SMC proliferation heightens reversible strokes)
synthesis of collagen, thereby hardening the
plaque 3. Aortic Atherosclerosis(aorta)
6. During the process, foam cells die releasing its - manifests as either
content. As the plaque grows, it builds in o aortic aneurysm (abnormal widening
pressure causing THROMBOSIS. of artery)
7. Thrombosis will cause blood coagulation o aortic dissection (tear in thoracic or
forming a clot, thus, impeding blood flow. abdominal)

4. Peripheral Artery Disease (PAD)

- disease of blood vessels supplying the arms
and legs
- manifests by
o intermittent claudication (acute
localized pain to the arms and legs)

5. Rheumatic Heart Disease

- Permanent damage to heart muscle, mainly
the valves, from the inflammation and
scarring caused by rheumatic fever
- Group A Strep = S. pyogenesis start as
tonsilitis, sore throat

6. Congenital Heart Disease

- malformations of heart structure or disorders
of the central blood vessels of the heart
existing at birth

Annie June Audan

 ENZYME CARDIAC MARKERS
- used in detecting cardiac disorders,
determining the risk of developing cardiac
disorders, monitoring the disorder or
predicting the response of a disorder to a
treatment
a. Rapidly released,
b. More specific (detect the specific analyte
without interference to other) and sensitive
(detect the smallest)
c. Persist in the circulation even in several days
- all enzyme are proteins, but not all proteins
are enzyme (troponin, myoglobin- protein but
not enzyme: AST, CK, LDH- protein and
enzyme)

ALL enzymes are proteins but not ALL proteins are

enzyme
2. Myoglobin
AST LDH CK-MB - An iron- and O2-binding protein exclusively
(More specific (More (Best found in the muscle; normally absent from the
than LDH) sensitive cardiac circulation
Aka Glutamic than AST) enzyme)
- First analyte to increase in AMI, half-life (9
Oxaloacetic Specific
mins), but not tested because short half life
Transaminase
- Cardiac and skeletal muscle; not specific
6-8 hours after 12-24 4-8
- Responsible of red coloration of the muscle
RISE onset of chest hours hours
o Muscle color depends on iron
pain of heart after after
oxidation state and amount of O2
attack onset of onset of
attached
chest chest
pain or pain or
3. B-Type Natriuretic Peptide BNP)
heart heart
- Cardiac stresses
attack attack
- Natriuretic peptides are secreted from the
PEAK 24 hours 48-72 12-24
heart in response to increased pressure and
hours hours
volume load
NORMAL 5 days 10 days 2-3 days
o Promotes: natriuresis, diuresis, and
vasodilation
OTHER CARDIAC MARKERS o Inhibits: sympathetic nervous system
1. Troponins signaling
- Complex of 3 proteins that regulate the
calcium-dependent interactions of myosin 4. Ischemia-Modified Albumin
heads with actin filament during striated - Related to oxidative damage during cardiac
muscle contraction ischemia
- Responsible for transmitting the calcium o Measures albumin change in the
signals that triggers muscle contraction presence of ischemia
o Troponin T (TnT): binds the troponin - Does not detect myocardial tissue damage
complex to tropomyosin - C-Reactive Protein
o Troponin I (TnI): inhibits the binding of
- (Inflammation, but not the location)
actin and myosin
- Non-specific
o Troponin C (TnC): binds to calcium to
reverse TnI function; expressed in
5. Heart-Type Fatty Acid Binding Protein (H-
slow-twitch (type 2) and cardiac
FABP)
muscle
- Similar to myoglobin, but more abundant to
- TnT and TnI are commonly used to assess
heart)
cardiac function; expressed in fast twitch (tyoe
- More sensitive but less sensitive than tnt
1), slow twitch, and cardiac muscle
- Not a routine test
- Gold standard test for myocardial infarction
- Best cardiac marker (general)
6. C-Reactive Protein
o Rise: 3-12 hours after onset
- Acute marker of inflammation
o Peak: 12-24 hours
o Inflammation is important in
o Normal: TnT: 8-21 days
development of atherosclerosis
TnI: 7-14 days
- Highly sensitive: increased 1000-folds

7. Homocysteine
- Increased amount is related to arterial
damage

Annie June Audan

 • AST (aspartate aminotransferase)
CARDIAC ENZYME DETERMINATION
Aspartate + a-Ketoglutarate → Oxaloacetate +
AST DETERMINATION Glutamate
ASPARTATE AMINOTRANSFERASE (AST)
- Aka Serum glutamic Oxaloacetic • MD (malate dehydrogenase)
transaminase (SGOT/ GOT)
- EC 2.6.1.1 L-Aspartate 2-oxaloglutarate Oxaloacetate + NADH + H → Malate + MAD
aminotransferase
- Widely used in human tissue
- Requires: pyridoxal-5-phosphate (P5P)
o helps catalyze the reaction by accepting
the amino group from the amino acid then
transferring it to the carbonyl compound

Significant tissue sources:

- Highest concentrations are found PREPARATION OF WORKING REAGENT
• Cardiac - Pipet 2 ml from the bottle SUB into another
• Liver bottle BUF, mix thoroughly
• Tissue and Skeletal muscle
- With smaller amount found in the PIPETTING SCHEME AND PROCEDURE
• Kidney 1. 0.02 mL Patient's Serum+ 1.0 mL Working
• Pancreas Reagent
• Erythrocytes 2. Mix and read at A1 after 1 minute
3. Read absorbance again after 1, 2 and 3
Notes minutes
➢ HEMOLYSIS should be avoided because it
dramatically increases AST CONCENTRATION
(𝐴1 − 𝐴2) + (𝐴2 − 𝐴3) + (𝐴3 − 𝐴4)
➢ AST is dramatically Increased in acute 𝐶𝑜𝑛𝑐 = 𝑋 952
hepatocellular disorders 3
➢ AST activity is stable in 3-4 days at
UNIT OF MEASURE
refrigerated temperature – 4 Celsius (not cold
- U/L
labile)
➢ AST used in AMI (acute myocardial Infarction)
CK-MB DETERMINATION
CREATININE KINASE – MB (CK-MB)
AST LEVEL DURING AMI:
- Aka CREATINE KINASE
Rise 6-8 hours
- EC 2.7.3.2
Peak 24 hours - ATP: Creatine N-phosphotransferase
Normal 5 days - Molecular weight: 82,000 daltons
- Generally associated with ATP regeneration in
REAGENT COMPOSITION contractile or transport systems
L-aspartate - Predominant in physiologic functions that
SUBSTRATE 2-oxoglutarate occurs in muscles cells
- Involved in the storage of high creatine
BUFFER TRIS buffer (pH 7.8) phosphate
MDH (malate
Creatine + ATP → Creatine phosphate + ADP
dehydrogenase)
- Every contraction cycle of muscle result in
ENZYME REAGENT
NADH creatine phosphate use, with the production of
(Nicotinamide ATP
adenine dinucleotide)
2 MAJOR SUBUNITS
- M and B
DIMERS
PRINCIPLE: CK-MM skeletal muscle
KARMEN method CK-MB cardiac tissue
- Coupled enzyme kinetic reaction CK-BB brain and intestine
- Reacts with aspartate and oxoglutarate to
form oxaloacetate and NADH Notes:
- Indicator: malate dehydrogenase (MDH) ➢ CK-MB are found in small quantity and other
- Measured at 340 nm tissue
- Change in absorbance is monitored as NADH ➢ Myocardium is essentially the only tissue from
is oxidized into NAD+ which CK- MB enters the serum in significant
quantities
➢ CK-MB – widely used in cardiac function level
determination

Annie June Audan

 ➢ Following the AMI (Acute Myocardial • CK
Infarction) the CK-MB levels will begin to
increase Creatine phosphate + ADPC → Creatine + ATP

CK-MB LEVELS DURING AMI • HK

Rise 4-8 hours
D-Glucose + ATP → HD-Glucose-6P + ADP
Peak 12-24 hours
Normal days • G-6PDH

- Increased quantities are not entirely specific D-Glucose-6-phosphate-NADP → D-Gluconate-

for AMI but probably reflects a degree of 6-P + NADPH + H
ischemic heart damage
- The specificity of CK-MB levels in the
diagnosis of AMI can be increased if
interpreted in conjunction with your lactate
dehydrogenase isoenzymes and tropidines if
measure sequentially over 48 hours period to
detect the typical rise and full of your enzymes
seen in AMI
- CK activity in serum is unstable (rapidly
inactivated because of oxidation of sulfhydryl
groups)
- Inactivation can be partially reversed by
addition of:
• N-acetylcysteine
• Mercaptoethanol
• Thioglycerol or dithiothreitol PREPARATION OF WORKING REAGENT
- Reconstitute one vial enzyme/antibody
REAGENT COMPOSITIONS reagent (ENZ) with 3 ml buffer
✓ ADP - Solution (BUF) Swird gently
✓ AMP - Incubate for 5 minutes at room temperature
✓ Diadenosine pentaphosphate before use
✓ NADP
ENZYME/ ✓ Creatine phosphate PIPETTING SCHEME AND PROCEDURE
ANTIBODY ✓ HK 1. 0.04 mL Patient's Serum +1.0 mL Working
Reagent
✓ G6PD
2. Mix and read at A1 after 10 minutes
✓ N-acetylcysteine
3. Read A2 after 5 minutes
✓ Antibody against CK-M
subunit CONCENTRATION
(𝐴2 − 𝐴1))
𝐶𝑜𝑛𝑐 = 𝑋 8254
Imidazole buffer (pH 6.7) 5
UNIT OF MEASURE
- anti-fungal - U/L
BUFFER
✓ Glucose
✓ Magnesium acetate LDH DETERMINATION
✓ EDTA LACTATE DEHYDROGENASE (LDH)
- EC 1.1.1.2.7 L – Lactate: NAD+
Oxidoreductase
- Catalyzes the interconversion of lactic and
PRINCIPLE pyruvic acids
COUPLED ENZYME KINETIC REACTION+ IMMUNO- - Hydrogen transfer enzymes uses
INHIBITION METHOD o Coenzyme: NAD+
- Antibody bind to the M subunit to inhibit its - widely distributed in the body
activity leaving the B subunit measurable - Non-specific but highly sensitive in
- Neglectable amounts of CK-BB (1%), inflammation and infections
remaining activity is multiplied by 2
(represents CK-MB activity) INCREASE LDH
HEMOLYSIS LDH is the first enzyme
increases

CHRONIC LDH increased because it

SMOKING also be found in the lungs
DISORDERS
CANCERS bone cancer, lung cancer
and liver cancer

Annie June Audan

Significant tissue sources: 3. Read absorbance again after 1, 2 and 3
- High activity minutes.
• Heart
• Liver CONCENTRATION
• Skeletal muscle
• Kidney (𝐴1 − 𝐴2) + (𝐴2 − 𝐴3) + (𝐴3 − 𝐴4)
𝐶𝑜𝑛𝑐 = 𝑋 8095
• Erythrocytes 3
UNIT OF MEASURE
- Found in lesser amount in - U/L
• Lung
• Smooth muscle
• Brain

MAJOR SUBUNITS
H and M
ISOENZYMES
HHHH (LD1) heart and RBCS
HHHM (LD2) heart and RBCs
HHMM (LD3) lymphoid and platelet
HMMM (LD4) liver and skeletal
MMMM (LD5) liver and skeletal

- Sera of HEALTHY INDIVIDUALS

o LD2 > LD1 > LD3 > LD4 > LD5
- Sera of PATIENTS WITH AMI (LD flip)
o LD1 > LD2 > LD3 > LD4 > LD5

LDH LEVELS DURING AMI

Rise 12-24 hours
Peak 48-72 hours
Normal 10 days

- LDH activity in serum is unstable (if not

processed immediately, store at 25°C &
analyze win 48 hours)
- Isoenzyme analysis store at 25°C & analyze
win 24 hours
✓ LD5: most heat labile isoenzyme
✓ LD1: most heat stable isoenzyme

PRINCIPLE
BACKWARD REACTION
- LDH catalyzes the conversion of pyruvate to
lactate using NADH as coenzyme
- Measured at 340 nm
- Rate of conversion of pyruvate to lactate and
of reduced NADH to oxidized NAD

REAGENT COMPOSITION
Enzyme reagent NADH
Substrate Pyruvate
Buffer Tris buffer (pH7.4)

PREPARATION OF WORKING REAGENT

- Pipet 2 mL from the bottle SUB into BUF, mix
thoroughly.

PIPETTING SCHEME AND PROCEDURE

1. 0.02 ml. Patient's Serum +1.0 mL Working
Reagent
2. Mix and read at A1 after 1 minute

Annie June Audan

 Food and Drug Administration (FDA)
TOXICOLOGY AND DRUGS OF ABUSE o
– oversees human safety issues
assoc. with therapeutic drugs,
cosmetic, food additives
- Toxicology is the study of the adverse effects
o Environmental Protection Agency
of xenobiotics in humans
(EPA) – regulates industry-related
- Xenobiotics refer to chemicals and drugs that
chemicals (pesticides, fungicides) that
are not normally found or produced in the
may threaten safe drinking water and
body. They are exogenous agents that have
clean air
adverse effects on living organisms. They
o Occupational Safety and Health
most often refer to environmental chemicals or
Administration (OSHA) – ensures
drug exposures such as antibiotics and anti-
safe and healthy work environments
depressants.
o Consumer Product Safety
- Poisons are also exogenous agents that have
Commission (CPSC) – regulates
an adverse effect on living organisms but
household chemicals
more often refers to those that are found or
o Department of Transportation (DOT)
caused by the environment such as animal,
– oversees transport of hazardous
plant, mineral or gas poisons.
chemicals
- Toxins are endogenous substances that are
biologically synthesized either in living cells or 4. Forensic toxicology
in microorganisms such as bacterial toxins.
- Medicolegal consequences of exposure to
- Acute toxicity refers to a single, short-term drugs or chemicals
exposure to a substance that is enough to
- Applied to drug testing
cause immediate toxic effects
- Major focus: establish and validate analytic
- Chronic toxicity refers to repeated or frequent
performance of test methods for legal
exposure at doses that do not cause
evidences (cause of death)
immediate toxic effects
o Accumulation of the toxicant or the
5. Clinical toxicology
toxic effects within the individual
- Studies the interrelationship between
o May affect different systems
xenobiotics and disease states
- Types of Toxins
o Venom - toxic secretion produced by
6. Environmental toxicology
animals typically delivered via
- Evaluation of environmental chemical
infliction of a wound, using spikes,
pollutants and their impact to human health
fangs, stain. Active.
- Monitor occupational health issues and
o Poison - Substances that are
increase public health monitoring
passively introduced to humans. Root
of exposure can be ingestion,
DOSE-RESPONSE RELATIONSHIP
inhalation, or absorbed in the skin
- “the dose makes the poison”
o any substance has the potential to
ROUTES OF TOXIN EXPOSURE
cause harm even fruits and water can
1. Ingestion (most common)
cause harm depending the amount or
2. Inhalation
degree of exposure
3. Transdermal absorption/Contact
o coined by Paracelsus (1493-1591)
- TD50 (Toxic Dose): dose that is predicted to
SCOPE OF TOXICOLOGY
produce a toxic response to 50% of the
1. Mechanistic toxicology
population
- Concerned with the dose-response
- LD50 (Lethal Dose): dose that would predict
relationship between the xenobiotic and the
death in 50% of the population
adverse effect
- ED50 (Effective Dose): dose that is predicted
o Elucidating the cellular, molecular,
to produce a therapeutic or effective response
and biochemical effects
to 50% of the population
- Involved in the development of tests to assess
- Therapeutic index: ratio of TD50 (or LD50) to
the degree of exposure
ED50
- Provides a basis for rational therapy deign
- The larger therapeutic index, the fewer toxic
adverse effects
2. Descriptive toxicology
- Individual dose-response relationship –
- Uses animal experiments to predict the
individual’s health status as well as changes
dosage level that can cause harm in human
in xenobiotic exposure level
(“risk assessment” process)
- Quantal dose-response relationship – the
change in health effects of a defined
3. Regulatory toxicology
population based on changes in the exposure
- Interpretation of data from mechanistic and
to the xenobiotic
descriptive toxicology to establish standards
of safety
- Works with or for government agencies

Annie June Audan

 Lethal Oral Dose in
Toxic Rating
Average Adult
Super Toxic <5 mg/kg
Extremely Toxic 5-50 mg/kg
Very Toxic 50-500 mg/kg
Moderately Toxic 0.5-5 g/kg
Slightly Toxic 5-15 g/kg
Practically Nontoxic >15 g/kg
ANALYSIS OF TOXIC AGENTS
- Includes
o routine testing – presence of a
number of agents might be present
(drug screens, heavy metal panels)
o targeted testing – performed when
an environmental risk of exposure is
known; support exposure
investigation; to comply with
occupational regulations or
guidelines; to confirm clinical
suspicions of poisoning
- Specimen
o Urine
• 24-hour collection – preferred to
compensate for variable
elimination patterns throughout
the day
• Random urine – not accurate;
screening and qualitative
detection of exposure
o Blood
• Royal blue top tube – for most
trace elements
• Tan top tube – lead
determinations
• DO NOT USE TUBE WITH GEL as
it may absorb the drugs
- Best specimen depends on the toxic agent’s
unique absorption, distribution, metabolism
and elimination kinetics
2-step procedure:
1. Screening test
- Rapid, simple, qualitative procedure
- Detect presence of specific substance or
classes of toxicants
- Good analytic sensitivity but lack specificity
- Negative result: rule out drug or toxic element
- Positive result: perform confirmatory test
2. Confirmatory test
- More specific method
- Quantitative and report the concentration of
substance present
Methods
- Thin Layer Chromatography
o Toxicology screening
o Qualitative (+,-)
o Simple and inexpensive method
o detects various drugs and other
organic compounds
- High Performance Liquid Chromatography –
o Quantitative measurement
o Able to identify the amount present
- stomach, and 80% will be absorbed by the intestine, go
- Gas Chromatography-Mass Spectrometry
o Gold standard test
o Reference test
o Quantitative ID of most organic
compounds
INTOXICATION - they are in a state of impaired mental
functioning resulting from ingestion of alcohol or drugs
or substances.
TOXIC AGENTS
Ethanol
- Grain alcohol
- Most commonly abused substance in the
world
- A CNS depressant
- Ethanol metabolic product: acetic acid
Alcohol→ alcohol dehydrogenase → Aldehyde →
aldehyde dehydrogenase → Acid
- Specimen: serum, plasma or whole blood
- Antiseptant should be alcohol-free; use
benzalkonium chloride
- Liver organ that is most affected
COMMON INDICATORS OF ETHANOL ABUSE
Test Comments
GGT Increases can be seen before the
onset of pathologic consequences.
Increases in serum activity can occur
in many non-ethanol-related
conditions.
AST Increases in serum activity can occur
in many non-ethanol-related
conditions.
AST/ALT A ratio of >2.0 is highly specific for
Ratio ethanol-related liver disease
HDL High serum HDL is specific for ethanol
consumption
MCV Increased in RBC MCV is commonly
seen with excessive ethanol
consumption.
Increases are not related to folate or
Vit B12 deficiency.
BLOOD
SIGNS AND SYMPTOMS
ALCOHOL
0.01-0.05 No obvious impairment, some
changes observable on performance
testing
0.03-0.012 Mild euphoria, decreased inhibitions,
motor skills impairment
0.09-0.25 Decreased inhibitions, loss of critical
judgment, memory impairment,
diminished reaction time
0.18-0.30 Mental confusion, dizziness, strongly
impaired motor skills (staggering,
slurred speech)
0.27-0.40 Unable to stand or walk, vomiting,
impaired consciousness
0.35-0.50 Coma and possible death
NOTES: 20% of the alcohol will be absorbed by the
Annie June Audan
to bloodstream, liver needs to degrade alcohol to be in
a lesser toxic form to be excreted because alcohol will
go to cells through passive diffusion. When alcohol
accumulates and suppresses brain. In the brain we have
neurotransmitter glutamine, and alcohol will suppress it
causing to be slower. You remember less, notice less.
Alcohol dehydrogenase will metabolize alcohol into
acetaldehyde. Acetaldehyde responsible for the
negative effects of alcohol in the body. It will be further
converted to acetic acid to be lesser toxic. If
acetaldehyde builds up, it will lead to loss of
coordination, memory problems, and sleepiness. When
alcohol leaves the system brain will compensate the
suppression leading to excitability and irritability. Brain
is awake will interrupt the sleep cycle. Makatulog pero
putol putol. As if you are sedated.
Pag walay kaon dali mahubog kay 20% ang ma absorb
sa stomach na alcohol. If nay kaon iabsorb sa food na
muabsorb sa alcohol. And since some will be absorbed
in the bloodstream, pass through the heart magka
tachycardia ang tao then muadto sa lungs. Mao ng nay
breath like sa alcohol anig human. Tas madistribute na
siya all throughout sa body. Possible maabot sa skin.
Manimahog alak pati ang sweats smells like alcohol.
Increase urination is because alcohol decreases ADH
levels. Hangover you felt, attributed to acetaldehyde
accumulationin the brain. Even dehydration and thirst.
Stomach pain caused by gastric acid. If acetaldehyde
mahimong acetic acid masuka nimo tanan kay acid.
Some are allergic due genetic factor. They lack enzymes
to degrade toxic alcohols. Gamayng inom kay mamula,
mangatol, and even asthma attack. Some people
paspas magconvert sa acetaldehyde but slow to
convert to acetic acid. Hubog dayon.
NO ANTIDOTE FOR ETHANOL TESTING.
Methanol
- Wood alcohol
- Seen in many commercial products or
contaminant of homemade liquors
- Metabolized by hepatic ADH to intermediate
formaldehyde
- Methanol metabolic product: formic acid
o Can case severe metabolic acidosis
w/c leads to tissue injury and death
o Responsible for optic neuropathy
leading to blindness
Less severe than ethanol
Isopropanol
- Rubbing alcohol
- Metabolic product: acetone
o has long half-life
- Has CNS depressant effect like ethanol
- Severe acute-phase ethanol like symptoms
that persist for an extended period
Ethylene Glycol
- Metabolic product: oxalic acid & glycolic acid
o Can lead to severe metabolic acidosis
- Presence of calcium oxalate crystals
(monohydrate/ whewellite) in urine
- Present in anti-freeze products and hydraulic
fluid
- Ingested accidentally or intentionally
o Common in children due to sweet
taste
Determination of Alcohol
- Specimens: serum, plasma, whole blood
- Serum has a higher concentration per unit
volume than whole blood
o Serum has greater water content
Ethanol Determination Considerations
1. Use alcohol-free disinfectant in cleaning the
venipuncture site.
2. Specimens must be capped at all times to
avoid evaporation.
3. Sealed specimens can be refrigerated or
stored at room temp for up to 14 days
without loss of ethanol.
4. Use sodium fluoride in preserving nonsterile
specimens or those intended for prolonged
storage to avoid increase in ethanol content
due to bacterial fermentation.
Analytic Methods for Ethanol Determination
1. Osmometric
- Measured by freezing point depression
-  serum osmolality =  serum ethanol conc
-  10mOsm/kg serum osmolality per 60
mg/dL in serum ethanol
- Osmolal gap = measured osmolality –
calculated osmolality
- Not ethanol specific
- Screening test
2. Gas Chromatography
- Reference method
- Quantitative
- Serum or blood sample is diluted with a
saturated solution of sodium chloride in a
closed container
3. Enzymatic
- Use a nonhuman form of ADH to oxidize
ethanol in the specimen to acetaldehyde
with simultaneous reduction of NAD+ to
NADH
- Absorbance at 340 nm
- Specific for ethanol
- Negative or low result may indicate
methanol or isopropanol intoxication
- Fully automated; not require specialized
instrumentation
Carbon Monoxide
- Produced by incomplete combustion of
carbon-containing substance
- Colorless, odorless and tasteless gas that is
rapidly absorbed into the blood from
inhalation
- Very toxic
- Can be acquired from gasoline, engines,
improperly ventilated furnaces, wood or
plastic fires
- Can cause cellular hypoxia because it
interferes with O2 transport
- Carbon monoxide + hemoglobin =
carboxyhemoglobin (COHb)
Annie June Audan
 - Binds to heme protein (hemoglobin)and has o Less harmful
200x-225x affinity for oxygen compared to o Found in seafood
normal hemoglobin o Rapidly absorbed by passive
- Shift to the left in the oxygen-dissociation diffusion in the GI tract
curve (low O2) o Cleared in urine within 48 hours
- Distinguishing characteristic: cherry red - Has high affinity binding to the thiol groups in
coloration of face & blood proteins
- Treatment: 100% oxygen therapy - Distinguishing characteristic: garlic breath
o For severe cases: hyperbaric chamber odor and metallic taste
- COHb half-life: 60-90 mins in people with - Specimen: urine (specimen of choice since it
normal respiratory function stays positive in the urine within 6 days),
keratinized parts such as hair and nails (to
COHb Symptoms & Comments assess long-term exposure; 2weeks)
0.5 Typical in nonsmokers - Mees lines: white line in fingernails
5-15 Range of values seen in smokers. - Analysis: Atomic Absorption Spectrometry
10 Shortness of breath with vigorous exercise (AAS)
20 Shortness of breath with moderate exercise
30 Severe headaches, fatigue, impairment of Cadmium
judgement - Found in paints, plastics and batteries;
40-50 Confusion, fainting on exertion tobacco-containing products
60-70 Unconsciousness, respiratory failure, death - Binds to proteins and accumulates in the
with continuous exposure kidneys (manifests with renal tubular
80 Immediately fatal dysfunction)
- Smoking double the lifetime body burden of
Cyanide cadmium
- Super toxic substance that exists as gas, solid, - Also causes parathyroid dysfunction and Vit D
or solution deficiency
- Hydrocyanic acid - Itai itai: disease characterized by severe
- Can be acquired from insecticides and osteomalacia and osteoporosis due to long-
rodenticides term cadmium-containing rice
- Binds to heme iron - Elimination is very slow
- Most common suicide/homicide agent - Biological half-life: 10 to 30 days
- Distinguishing characteristic: odor of bitter - Analysis: AAS used in whole blood or urinary
almonds breath content
- If inhaled, rapidly absorbed in alveolar
Lead
capillaries
- Rapid onset of symptoms - Found in paint
o Related to cellular hypoxia - Can cause mental retardation,
o Headache encephalopathy, combulsions, impaired
o Flushing consciousness
o Dizziness - Most common exposure: ingestion of
o Respiratory depression contaminated dietary constituents
- It can progress to coma, seizure, heart - Distributes in 2 compartments
blockage, death o Bones
▪ Combines to matrix of bones
Arsenic ▪ half-life 20 years
- A metalloid o Soft tissues
- Can cross the placenta ▪ Half-life: 120 days
- Common homicide and suicide agent - Potent inhibitor of many enzymes
o Affects Vit D metabolism and heme
- Toxicity depends on valence state, solubility,
synthetic pathway
and rate of absorption and elimination
- Causes anemia (presence of basophilic
- 3 major groups for arsenic
stippling)
1. Arsine gas
o Arsine trioxide - Causes kidney problems
o Inhalation: most acute toxicity - Characteristics: wrist drop/foot drop
2. Inorganic form manifestation
o Trivalent and pentavalent - Specimen: whole blood or urine (recent
o Absorbed at a slower rate in the GI exposure)
tract - Occupational exposure who works in battery
o Initial half-life: 10 hours factories
o 70% is secreted in urine where 50% - Treatment:
excreted is transformed to organic o Cessation of exposure
form o Therapeutic chelators (EDTA and
3. Organic forms dimercaptosuccinic acid) which
o Arsenobetaine and Arsenocholine removes lead from soft tissue and
o Fish arsenic bone

Annie June Audan

 COMPARISON OF EFFECTS OF LEAD ON CHILDREN - Results in excess ketone body formation due
AND ADULTS to stimulation of mobilization and use of free
fatty acid
- Treatment: neutralize and eliminate excess
acid; maintain electrolyte balance
- Analysis:
o GC and Liquid chromatography –
sensitive and specific
o Trinder reaction – chromogenic assay

DRUGS OF ABUSE
- clinical drugs being abused

Amphetamines
- Shabu (metamphetamine HCl)
- Treatment for narcolepsy and attention deficit
disorder (ADHD)
- Stimulant; increases mental alertness (uppers)
- Methylenedioxymethylamphetamine
(MDMA)/Ecstasy
o Designer drug; derivative of
metamphetamine
o Oral administration (50-150 mg)
o Half-life: 8-9 hrs.
o Onset of effect: 30-60 mins
Mercury o Duration: 3.5 hrs.
- 3 forms:
Anabolic steroids
o Elemental: largely not absorbed;
liquid at room temp - Chemically related to testosterone
o Inorganic salts: partially absorbed but - Used as therapy for male hypogonadism
can still cause GI toxicity - Increases muscle mass, power, stamina, vigor
o Organic compounds: rapidly and strength
absorbed through passive diffusion - May lead to immune suppression
- Found in thermometers; already toxic when - Commonly abused drug by athletes
inhaled - Side effects: increase muscle mass
- Common reason: inhalation and accidental o Males: testicular atrophy, sterility, and
ingestion impotence
- Major source of exposure: consumption of o Females: development of masculine
contaminated food traits, breast reduction, sterility
- Different toxic characteristics
Cannabinoids
o Elemental mercury (Hg0) – ingested
- Marijuana/Hashish
w/o significant effects
o Cationic mercury (Hg2+) – moderately - Smoked or ingested
toxic - Urinary Metabolite: THC-COOH (11-nor-
o Organic mercury (methylmercury; deltatetrahydrocannabinol)
CH3Hg+) – extremely toxic - (+) in urine up to 45 days
- Inhibits enzymes o 1st time user – 2-5 days (+)
- Causes kidney problems o Chronic smoker – 3-4 weeks (+)
- Most toxic effect: Pink disease/ acrodynia - Tetrahydrocannabinol (TCH)
o Most potent and abundant
- Analysis: AAS using whole blood or an aliquot
o Lipophilic substance – rapidly
of 24-hour urine or anodal voltammetry
removed from circulation by passive
distribution into hydrophobic
Salicylates
compartments (brain, fat)
- acetylsalicylates or aspirin
- Half-life: 1 days (1st time user); 3-5 days
- analgesic, antipyretic, and anti-inflammatory
(chain smoker)
drug
- Causes loss of intellectual function, poor
o decreases thromboxane and
memory and reddening of conjunctiva
prostaglandin formation
- Cannabis is the plant
- associated with non-respiratory acidosis
- Most frequently used drug
- direct stimulator of the respiratory center
- It can treat anorexia, nausea
- inhibits Krebs cycle leads to accumulation of
lactic acid

Annie June Audan

Cocaine REVIEW ON DRUG TESTING
- Crack - Screening specimen: random urine
- Local anesthetic - Best specimen: urine (30-60 ml)
- Half-life: 30 mins to 1 hour - Other specimens: 10 ml blood, 30 ml saliva
- Metabolite: benzoylecgonine - Temperature: 32-38°C tested within 4 minutes
o Half-life: 4-7 hrs. - Urine specific gravity: 1.003-1.035
o Detected in urine for 3 days after - Urine creatinine: < 20 mg/dl
single use - Adulterants: substances added to urine that
o 20 days for chronic heavy abuser will cause false negative results
o Measured for primary screening test - Water is the most common adultering agent.
for cocaine use Salt, vinegar, baking soda, liquid soap, bleach
- Can cross the placenta and mammary gland can also be used as adulterants
- Not a true addictive drug - Positive urine test- average of 12 hours to 3
- Mental alertness weeks
- Recreational drug
- Administration
o Direct
▪ Insufflation/ intravenous injection
o Inhalation
▪ As a vapor when smoked in the
free base form (crack)

Opiates
- Analgesic, sedative and anesthetic
- Natural forms:
o Opium,
o Morphine,
o Codeine
- Chemically modified from natural form
o Heroin
o Hydromorphone (Dilaudid)
o Oxycodone (Percodan
- Synthetic forms:
o Meperidine (Demerol)
o Methadone (Dolophine)
o Propoxyphene (Darvon)
o Pentazocine (Talwin)
o Fentanyl (Sublimaze)
- Toxic effect: pin point pupils

Phencyclidine
- Angel dust or Angel hair
- Stimulant, depressant, anesthetic, and
hallucinogenic
- Administration: Ingestion or Inhalation of PCP-
laced tobacco or marijuana
- 10-15% is eliminated and unchanged in urine
- In heavy users, can be detected up to 30 days
after abstinence
- MOT: isolation
- Dissociative drug
o Dissociation of pain, movement
- Potent veterinary analgesic and anesthetic

Lysiergic Acid Diethylamide (LAD)

- Most potent drug (20μg)
- Causes “bad trip” or sudden change of mood
- Toxic effect: blurred/undulating vision

Tryptamines
- Causes sudden hallucinations
- DMT aka “businessman’s lunch”
- Psilocycin aka “magic mushroom”

Annie June Audan

 THERAPEUTIC DRUG MONITORING CLASS DRUGS
Digoxine,
- Therapeutic Drug Monitoring (TDM) involves Cardioactive Lidocaine,
the analysis, assessment and evaluation of Propanolol
circulating concentrations of drugs in serum, Lithium (treatment for manic
plasma or whole blood depression, bipolar disorders)
Anti-depressants
Prozac (attempted suicide
Purpose of TDM: patients)
- Ensure that a given drug dosage is within a Acetylsalicylic acid,
range that produces maximal therapeutic Acetaminophen (lead to
benefit Anti-inflammatory
hepatotoxicity when
- Identify when the drug is above or below overdosed)
therapeutic range Valproic acid (absence
Anti-convulsants seizures/petit mal & grand
Indications of TDM: mal)
- Identify non-compliance of patients
Cyclosporine,
- Prevent overdosing or underdosing Immunosuppresive
Tacrolimus
- Maximizing therapeutic effect Methotrexate,
- Optimizing a dosing regimen Anti-neoplastic
Busulfan
Bronchodilators Theophylline
Routes of Administration
Aminoglycosides,
1. Intravenous: most direct route Vancomycin,
2. Intramuscular: injected directly into the Antibiotics
Chloramphenicol,
muscles Trimethoprim lactate
3. Subcutaneous: injected under the skin
4. Transcutaneous: inhaled or absorbed through
the skin
5. Suppository: rectal delivery
6. Oral: most common route

Specimen Considerations
- Timing of Specimen is the single, most
important factor
- Trough concentrations
o Lowest concentration of drug in the
body
o Collected right before the next dose
- Peak concentrations
o Highest concentration of drug in the
body
o Orally administered: collected 1 hour
after (except digoxin)
o Intravenously administered: collected
30 min after
- Specimen of choice: serum or heparinized
plasma (best)
- Refrain from using gel or serum separator
tubes as some drugs can be absorbed in the
gel

Annie June Audan

BASIC PRINCIPLES & PRACTICES 103 Kilo k 1,000.0
- The purpose of a clinical chemistry laboratory is to 106 Mega M -
facilitate the correct performance of analytic 109 Giga G -
procedures that yield accurate and precise 1012 tera T -
information, aiding patient diagnosis and 1015 Peta P -
treatment. 1018 axa E -
- The achievement of reliable results requires that
the clin laboratory scientist be able to correctly use REAGENTS
basic supplies and equipment and possess an - A compound r mixture added to a system to
understanding of fundamental concepts critical to cause a chemical reaction or test if a reaction
any analytic procedure. occurs
- May be used to tell whether or not a specific
UNITS OF MEASUREMENT chemical substance is present by causing a
Quantitative laboratory results: reaction to occur with it.
1. Number related to the actual test value
2. Label identifying unit CLINICAL LABORATORY SUPPLIES
Ex. FBS = 5.5 mmol/L 1. Thermometers/temperature
- The predominant practice for temperature
SYSTEM INTERNATIONAL (SI UNITS) measurement uses the Celsius (°C) scale;
- It was adopted internationally in 1960, is however, Fahrenheit (°F) and Kelvin (°K) scales
preferred in scientific literature and clinical are also used. The SI designation for
laboratories and is the only system employed temperature is the Kelvin scale
in many countries. Three major types of thermometer
- This system was devised to provide the global o Liquid – in – glass: 20-400 degree
scientific community with a uniform method of Celsius
describing physical quantities. ▪ Partial immersion
BASE QUANTITY Name Symbol thermometer – water bath
Length Meter m ▪ Total immersion
Mass Kilogram Kg thermometer – refrigeration
Time Second s application
Electric current Ampere A o Electronic thermometer (thermistor
Thermodynamic Kelvin K probe)
temperature o Digital thermometer
Amount of substance Mole mol
Luminous intensity Candela cd 2. Glassware
SELECTED DERIVED o Borosilicate Glass
Frequency Hertz Hz ▪ Most common type of
Force Newton N volumetric measurement
Celsius temperature Degree Celsius °C ▪ A sodium-aluminum
Catalytic activity Katal Kat borosilicate glass with
excess of silica
SELECTED DERIVED NON- SI
▪ High degree of thermal
Minute (time) (60 s) Min
resistance
Hour (3600 s) H ▪ Low alkali content, and is
Day (86400 s) D free from magnesium-lime-
Liter (volume) (1dm3=10- L zinc group of elements,
3m3) heavy metals, arsenic and
Angstrom (0.1 nm=10- A antimony
10m) ✓ Pyrex (resistance of up
to 515 degree C)
SELECT ✓ Kimax
FACTOR PREFIX SYMBOL
DECIMAL o Alumina – silicate (Aluminosilicate)
10-18 atto a Glass
o ▪ Glass that are strengthened
chemically rather thermally
10-15 Femto f - ▪ 6x stronger than borosilicate
10-12 Pico p - glass
10-9 Nano n - ▪ Will outcast any
10-6 Micro μ 0.000001 conventional glasswares by
10-3 Milli m 0.001 10-fold
10-2 Centi c 0.01 ▪ Resist clouding and
10-1 Deci d 0.1 scratching better
100 Liter, Basic unit 1.0 ✓ Corex Brand
meter,gram o Low Actinic Glassware
101 Deka da 10.0 ▪ High thermal resistance
102 Hector h 100.0 glass

Annie June Audan

 ▪ Has an amber or red color
added as an integral part of
the glass, in order to protect
light-sensitive material
(bilirubin standards), yet
permits visibility of contents
▪ For containers used to store
control materials and
reagents
3. Plasticware
o Polypropylene
▪ Flexible, chemical resistant,
can be autoclaved
▪ Used as piper tips and can
withstand below -190
degree C
o Polyethylene
▪ Widelt used in test tubes,
bottles, graduated tubes,
stopers, disposable transfer
pipets, volumetric pipets, and
test tube racks
▪ May absorb proteins, dyes,
stains, and picric acids
o Polycarbonate
▪ Very strong plastic with
usable temperature range of
-100 to +160 degree C and
can be autoclaved
▪ Not suitable for strong acids,
bases, and oxidizing agents
o Polystyrene
▪ Rigid, clear type of plastic
used in capped graduated
tubes, will crack and splinter
when crushed
o Teflon
▪ Almost chemically inert and
is suitable for use at
temperature -270 to +255
degree C. Widely used for
stirring bars, tubing,
cryogenic vial and bottle cap
liners
4. Laboratory Vessels
a. Flasks
o Volumetric Flask
▪ calibrated to hold one exact
volume of liquid (TC)
▪ Has a round, lower portion
with flat bottom and a long
thin neck with an etched
calibration line
o Erlenmeyer Flask
▪ Designed to hold different
volumes rather than one
exact amount
b. Beakers
c. Graduated cylinders
▪ Long, cylindrical tubes
usually held upright by an
octagonal or circular base
5. Pipets
- Glass or plastic utensils used to transfer
liquids, they may be reusable or disposable.
Although pipets may transfer any volume,
they are usually used for volumes of 20 mL or
less.
I. Design
a. To Contain – holds or contains a
particular volume but does not
dispense that exact volume
b. To Deliver – will dispense the volume
indicated
II. Drainage characteristics
a. Blowout – has etched colored rings
b. Self – draining
III. Type
a. Measuring or graduated
i. Serologic
ii. Mohr
iii. Bacteriologic
iv. Ball, Kolmer, Kahn
v. Micropipet
b. Transfer
i. Volumetric
ii. Ostwald-folin
iii. Pasteur pipets
iv. Automatic
micropipettes/micropipettes
6. Burets
- Looks like a wide, long, graduated pipet with a
stopcock at one end
- A burets usual total volume ranges from 25 to
100 ml of soln
- Used to dispense a particular volume of liquid
during titration
7. Syringes
BASIC SEPARATION TECHNIQUES
1. Centrifugation
- a process in which centrifugal force is used to
separate solid matter from a liquid
suspension.
- It is used to prepare samples, blood and body
fluids, in clinical chemistry for analysis and
also to concentrate urine sediment in
urinalysis for microscopic viewing.
- Centrifugal force depends on three variable:
mass, speed, and radius.
- The speed is expressed in revolutions per
minute (rpm), and the centrifugal force
generated is in terms of relative centrifugal
force (RCF) or gravities (g). The speed of the
centrifuge is related to the CRF
- Rounds per minute (RPM) of the centrifuge is
calibrated by TACHOMETER
- Centrifuge machine should be disinfected
every week and calibrated every 3 months
SWINGING BUCKET CENTRIFUGE
- Horizontal Head Centrifuge
- Centrifuge tubes are held in a vertical position
when not moving but are horizontal when in
full motion
- Generate low speeds only but can produce a
tight pellet of precipitate or clotted cells in the
bottom of the tube
Annie June Audan
FIXED HEAD CENTRIFUGE - Remember that 1 mol of substance is equal to
- Has a fixed 25 – 52 degree angle at w/c the the gmw (gram molecular weight) of that
tubes are held during centrifugation substance.
- The sediment packs at an angle but not as
tightly as with a horizontal had centri 5. Dilutions
- A dilution represents the ratio of concentrated
ULTRACENTRIFUGATION or stock material to the total final volume of a
- Generates the highest speed; in order to solution and consists of the volume or weight
reduce the heat produced by the friction of the concentrate plus the volume of the
generated by high centrifugal speed, diluent, with the concentration units remaining
ultracentris are refrigerated the same.
- This type of centri is especially useful for - 2 forms of dilution
lipoprotein since refrigeration enhances the o Simple dilution
separation o Serial dilution
2. Filtration
3. Dialysis
- The dialysis machine mixes and monitors the
dialysate.
- Dialysate is the fluid that helps remove the
unwanted waste products from the blood. It
also helps get the electrolytes and minerals to
their proper levels in the body.

LABORATORY MATHEMATICS
1. Unit Conversion
2. Percent Solution
- A percent solution is determined in the same
manner regardless of whether weight/weight,
volume/volume, or weight/volume units are
used. Percent implies “parts per 100,” which is
represented as percent (%) and is
independent of the molecular weight of a
substance.
o Example: W/W
To make up 250 g of a 5% aqueous
solution of hydrochloric acid (using 12 M
HCl), multiply the total amount by the
percent expressed as a decimal.
o Example: W/V
The most frequently used term for a
percent solution is weight per volume,
which is often expressed as grams per
100 mL of the diluent. How many grams
of NaOH needed to make a 1,000mL of
10% (w/v)soln?

3. Normality
- Often used in chemical titrations and chemical
reagent classification. It is defined as the
number of gram equivalent weighs per 1 L of
soln.
- Normality (N) is expressed as the number of
equivalent weights per liter (Eq/L) or
milliequivalents per milliliter (mmol/mL).
- Equivalent weight is equal to gmw divided by
the valence (V).

4. Molarity
- routinely expressed in units of moles per liter
(mol/L) or sometimes millimoles per milliliter
(mmol/mL).
- the SI representation for the traditional molar
concentration is moles of solute per volume of
solution, with the volume of the solution given
in liters

Annie June Audan